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1,861 | https://bio-protocol.org/exchange/protocoldetail?id=1861&type=0 | # Bio-Protocol Content
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Peer-reviewed
Subchromoplast Fractionation Protocol for Different Solanaceae Fruit Species
Marilise Nogueira
Harriet Berry
Rebecca Nohl
Martin Klompmaker
Alexandra Holden
Paul D. Fraser
Published: Vol 6, Iss 13, Jul 5, 2016
DOI: 10.21769/BioProtoc.1861 Views: 8899
Edited by: Ru Zhang
Reviewed by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Nov 2013
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The authors used this protocol in:
Nov 2013
Abstract
Macromolecules, proteins, lipids, and other small molecules, such as carotenoids can be studied within different tissues and organelles using an array of in vitro and in vivo methodologies. In the case of tomato and other fleshy fruit the predominant organelle in ripe fruit is the chromoplast. The characteristic feature of this organelle is the presence of pigments, carotenoids at high levels. In order to fully understand the underlying biological mechanisms that operate within the chromoplast, it is necessary to perform studies at the subchromoplast level. This protocol allows the separation of plastoglobules (lipoprotein particles, which are coupled to thylakoid membranes in the chloroplasts) and membranes (thylakoid, envelope-like) of chromoplasts through a sucrose gradient. The subchromoplast compartments can then be analysed independently. Comparisons between mutant/transgenic genotypes and their backgrounds can be performed accurately with simultaneous processing during the same fractionation run. The procedure was initially developed for ripening tomato fruit but translation to sweet and hot pepper has been shown.
Materials and Reagents
50 ml Falcon tubes (Sigma-Aldrich, catalog number: Greiner227261 )
Muslin (MacCulloch & Wallis, catalog number: 4470 )
Plants
Tomato (90 to 150 g of breaker +3 to 5 days fruit per condition)
Sweet bell pepper (30 to 120 g of ripe fruit per condition; depending on fruit pigment content)
Hot chilli pepper (~30 g of ripe fruit per condition)
Note: Breaker implies the first visual appearance of yellow/orange colour on the fruit. It is usually difficult to get ~10 tomato fruits (for 90 to 150 g total) at the same ripening stage on the same day, so that is why we harvest fruit from breaker + 3 days to breaker + 5 days. Of course, if you can have enough fruit at the same ripening stage, it is even better.
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503 )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Tricine (Sigma-Aldrich, catalog number: T0377 )
Sodium bisulphite (sodium metabisulfite) (Sigma-Aldrich, catalog number: S9000 )
Extraction buffer (see Recipes)
Gradient buffers (see Recipes)
Equipment
Cold room (4 °C)
-20 °C room
Centrifuge Sorvall RC-5C (Thermo Scientific)
Fixed angle rotor Sorvall GSA-3 (purple colour) (Thermo Scientific)
Fixed angle rotor Sorvall GSA-5 (green colour) (Thermo Scientific)
Ultracentrifuge Beckman L7 (Beckman Coulter)
Swing rotor SW28 (Beckman Coulter)
500 ml centrifuge bottles (Thermo Fisher Scientific, NalgeneTM, model: 3141-0500 )
50 ml centrifuge tubes (Thick-wall UltraTubes) (Thermo Fisher Scientific, NalgeneTM, model: 3110-0500 )
38.5 ml centrifuge tubes (Beckman Coulter, Ultra-ClearTM, model: 344058 )
Glass flask conical narrow neck 5 L (Fischer Scientific, FisherbrandTM, model: 11597422 )
Note: This product has been withdrawn by the manufacturer. [Replacement item: Borosilicate glass narrow neck Erlenmeyer flasks (Fischer Scientific, FisherbrandTM, model: 15479103 )]
Waring blender (small laboratory blender 8010ES) (Scientific Labs, model: MIX1126 )
Potter-Elvehjem tissue grinder (VWR International, model: 14231-372 )
Fraction collector (Gilson, model: 203B )
Suitable ice containers
Funnel (glass or plastic, to fit 5 L conical flasks)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nogueira, M., Berry, H., Nohl, R., Klompmaker, M., Holden, A. and Fraser, P. D. (2016). Subchromoplast Fractionation Protocol for Different Solanaceae Fruit Species. Bio-protocol 6(13): e1861. DOI: 10.21769/BioProtoc.1861.
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Category
Cell Biology > Organelle isolation > Chromoplast
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1,862 | https://bio-protocol.org/exchange/protocoldetail?id=1862&type=0 | # Bio-Protocol Content
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T Cell Transfer Model of Colitis
WP Wen Pan
SZ Shu Zhu
DD Dai Dai
YT Yuanjia Tang
YY Yihong Yao
Nan Shen
Published: Vol 6, Iss 13, Jul 5, 2016
DOI: 10.21769/BioProtoc.1862 Views: 17604
Edited by: Ivan Zanoni
Original Research Article:
The authors used this protocol in May 2015
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May 2015
Abstract
Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC) is mainly caused by disordered immune regulation and dysregulated intestinal microbiota. Here we present the T cell transfer model which has extensively used in many studies to identify the regulatory T cell function in gut inflammation. Naïve T cells only or together with Treg cells isolated from different donors were transferred into immunodeficient Rag1-/- mice and the disease progression was assessed by the loss of body weight and the scoring analysis. This model provides a reliable work system for the study of gut inflammation.
Keywords: Induced colitis Mouse model T cells Gut inflammation Rag1-/-
Materials and Reagents
15 ml polypropylene conical tubes (Corning, catalog number: 430790 )
50 ml polypropylene conical tubes (Corning, catalog number: 430828 )
LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
6-8 weeks old C57BL/6J WT mice used for donors (males)
6-8 weeks old Immunodeficient RAG1−/− mice (males) (The Jackson Laboratory, Stock number: 00 2216 )
10 cm petri dish (Corning, catalog number: 430167 )
10 ml syringe (BD Biosciences, catalog number: 301604 )
27 gauge needle (BD Biosciences, catalog number: 305109 )
40 μm nylon cell strainer (Corning, Falcon®, catalog number: 352340 )
70 μm cell filter (Corning, Falcon®, catalog number: 352350 )
Polystyrene round-bottom flow tubes (Corning, Falcon®, catalog number: 352054 )
FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10100-147 )
RPMI media 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093 )
ACK lysing buffer for erythrocyte lysis (Thermo Fisher Scientific, GibcoTM, catalog number: A10492-1 )
Mouse CD4 Negative Isolation Kit (Miltenyi Biotec, catalog number: 130-095-248 )
Fluorescently labeled antibodies:
anti-CD25-PE (BioLegend, catalog number: 102012 )
anti-CD4-PE-Cy7 (BD Biosciences, catalog number: 563933 )
anti-CD44-APC (BD Biosciences, catalog number: 559250 )
anti-CD62L-FITC (BD Biosciences, catalog number: 553150 )
37% Formaldehyde (formalin) Solution (Avantor Performance Materials, J.T.Baker®, catalog number: 2016 )
FACS buffer (see Recipes)
Equipment
Plain glass microscope slides (Omano, catalog number: OMSK-50PL )
Hemocytometer
Light microscope
Top-loading balance
Centrifuge
Flow cytometry (BD Biosciences, model: ARIA III )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pan, W., Zhu, S., Dai, D., Tang, Y., Yao, Y. and Shen, N. (2016). T Cell Transfer Model of Colitis. Bio-protocol 6(13): e1862. DOI: 10.21769/BioProtoc.1862.
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Category
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Cell imaging > Fixed-tissue imaging
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1,863 | https://bio-protocol.org/exchange/protocoldetail?id=1863&type=0 | # Bio-Protocol Content
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An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity
Raviraj M. Kalunke
Ilaria Moscetti
Silvio Tundo
Renato D’Ovidio
Published: Vol 6, Iss 13, Jul 5, 2016
DOI: 10.21769/BioProtoc.1863 Views: 8381
Edited by: Zhaohui Liu
Reviewed by: Igor Cesarino
Original Research Article:
The authors used this protocol in Aug 2015
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The authors used this protocol in:
Aug 2015
Abstract
Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present considerable industrial interest for their use in paper manufacturing, improvement of animal feed digestibility, and clarification of fruit juices. In addition, this enzyme is the component of cell wall-degrading enzymes (CWDEs) during plant–pathogen interaction. Thus, considering their various applications in plant defence and also in industry, the characterization of xylanase activity becomes an important aspect. Conventionally, xylanase activity is determined by radial gel diffusion assay using Congo red staining (Emami and Hack, 2001) and by DNSA assay which is a colorimetric method for xylanase activity (McLauchlan et al., 1999; Kutasi et al., 2001). Comparatively, radial gel diffusion assay using Congo red staining is a qualitative assay whereas DNSA method is a quantitative assay. Moreover, Congo red is a chemical considered as hazardous category 1B (Carcinogenicity) and category 12 (Reproductive toxicity) by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). In the present study, the proposed method enables qualitative detection of xylanase activity using ethanol precipitation in the radial gel diffusion assay which is safer and simpler. The ethanol precipitation in agar plate has been adapted from the method for detecting xylanase activity in polyacrylamide gels (Royer and Nakas, 1990).
Keywords: Xylanase Radial gel diffusion Xylan
Materials and Reagents
90 x 15 mm Petri plates (SARSTEDT AG, catalog number: 82.1473001 )
0.5-cm-diameter drinking straw
150 ml Erlenmeyer flask
Aspergillus niger xylanase (Xylanase M4) (Megazyme) or Trichoderma longibrachiatum xylanase (Xylanase M3) (Megazyme)
Disodium hydrogen phosphate (Sigma-Aldrich, catalog number: S5136 )
Citric acid (Sigma-Aldrich, catalog number: C1909 )
Birch wood xylan (Sigma-Aldrich, catalog number: X0502 )
Agarose (AppliChem GmbH, catalog number: A8963.0500 )
Absolute ethanol (VWR International, catalog number: 20821.321 )
McIlvaine’s buffer (pH 5.0) (see Recipes)
95% (v/v) ethanol (see Recipes)
Equipment
Microwave
Hallow flat end with 0.5-cm-diameter drinking straw
30 °C incubator (Mini BATT 805) (230 V, 50 Hz, 270 W) (Asal Srl, model: 805 )
Scanner camera (Epson, model: perfection V30 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kalunke, R. M., Moscetti, I., Tundo, S. and D’Ovidio, R. (2016). An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity. Bio-protocol 6(13): e1863. DOI: 10.21769/BioProtoc.1863.
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Category
Biochemistry > Protein > Activity
Microbiology > Microbial biochemistry > Protein
Plant Science > Plant immunity > Host-microbe interactions
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1,864 | https://bio-protocol.org/exchange/protocoldetail?id=1864&type=0 | # Bio-Protocol Content
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Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii
Takashi Yamano
HF Hideya Fukuzawa
Published: Vol 6, Iss 13, Jul 5, 2016
DOI: 10.21769/BioProtoc.1864 Views: 12075
Edited by: Maria Sinetova
Reviewed by: Cindy Ast
Original Research Article:
The authors used this protocol in Jun 2015
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Abstract
Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO2-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et al., 2015), and LCIA (chloroplast envelope anion channel; Yamano et al., 2015) in Chlamydomonas reinhardtii. The protocol described here could be useful for observing the protein of interest in other algae cells.
Keywords: Chloroplast membrane Bicarbonate transporter Micro-algae Photosynthesis Pyrenoid
Materials and Reagents
Poly-L-lysine-treated glass slide (Poly-Prep Slides) (Sigma-Aldrich, catalog number: P0425-72EA )
Coverslips (Matsunami Glass, catalog number: C218181 )
Dako Pen (Dako, catalog number: S2002 )
Plastic box (9 cm x 19 cm x 4 cm)
Note: In this experiment, but any size is OK.
Kimwipes® paper (Kimberly Clark)
Chlamydomonas cells of interest
Hematocytometer (Erma, catalog number: 03-303-6 )
Phosphate-buffered Saline (PBS)
Tween-20 (Santa Cruz, catalog number: sc-29113 )
PBS-T (PBS with 0.1% Tween-20)
Paraformaldehyde (Nacalai Tesque, catalog number: 26126-54 )
Methanol (Nacalai Tesque, catalog number: 21915-64 )
Globulin-free Bovine Serum Albumin (BSA) (Nacalai Tesque, catalog number: 01281-26 )
Affinity-purified rabbit primary antibody against LCIB (Yamano et al., 2010)
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Thermo Fisher, catalog number: A-11001 )
Mounting medium (Vectorshield, catalog number: H-1300 )
4% formaldehyde (see Recipes)
PBS (see Recipes)
PBS-T (see Recipes)
Blocking solution (see Recipes)
Equipment
Coplin jar (50 ml size)
Fluorescence microscopy Axioscope2 (Zeiss) with a specific filter set (excitation bandpass
480/40 and emission band pass 527/30) or laser scanning confocal microscopy TCS SP8
(Leica) with a 488 nm laser line
Note: these were the instruments used throughout our experiments, but any company is OK.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yamano, T. and Fukuzawa, H. (2016). Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii. Bio-protocol 6(13): e1864. DOI: 10.21769/BioProtoc.1864.
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Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Immunodetection
Cell Biology > Cell imaging > Fluorescence
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1,865 | https://bio-protocol.org/exchange/protocoldetail?id=1865&type=0 | # Bio-Protocol Content
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Denervation of Mouse Lower Hind Limb by Sciatic and Femoral Nerve Transection
YR Yuval Rinkevich
DM Daniel T. Montoro
EM Ethan Muhonen
DL David Lo
MH Masakazu Hasegawa
CM Clement D. Marshall
GW Graham G. Walmsley
Andrew Connolly
IW Irving L. Weissman
ML Michael T. Longaker
Published: Vol 6, Iss 13, Jul 5, 2016
DOI: 10.21769/BioProtoc.1865 Views: 15855
Edited by: Oneil G. Bhalala
Reviewed by: Antoine de Morree
Original Research Article:
The authors used this protocol in Jul 2014
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The authors used this protocol in:
Jul 2014
Abstract
The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. Reports from salamander models of appendage regeneration (Singer, 1952; Singer, 1947; Kumar et al., 2007), and of human clinical skin and nail problems associated with spinal cord injury patients (Stover et al., 1994) suggest that appendage regeneration may have an important nerve component. To explore this question, we have generated hind limb tissues devoid of nerve supply. This protocol, combined with multi-color ‘Rainbow’ reporter mouse lines permits single cell clonal analysis and genetic lineage tracing studies in the absence of nerve supply (Rinkevich et al., 2014), exposing nerve requirements on cellular replacement and differentiation during tissue growth, maintenance, and regeneration.
Keywords: Denervation Peripheral Nervous System Tissue Maintenance Limb Regeneration
Materials and Reagents
Gauze pad
Sterile drapes
4-0 plain gut suture, 18”, PS-4 needle (Ethicon)
Handheld cautery: high temperature cautery fine tip (Bovie Medical, model: AA01 )
Wild-type CD-1 mice (8-12 weeks old) (Charles River Laboratories International)
Isoflurane (isothesia) (Henry Schein Animal Health)
Veterinary ointment (Dechra, Puralube® Ophthalmic Ointment)
10% povidone iodine prep solution (Dynarex, catalog number: 1413 )
0.3 mg/ml buprenorphine (Buprenex) (Reckitt Benckiser)
Equipment
Electric hair clippers
Scissors
CO2 chamber
Far infrared warming pad (Kent Scientific)
Procedure
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How to cite：Rinkevich, Y., Montoro, D. T., Muhonen, E., Lo, D., Hasegawa, M., Marshall, C. D., Walmsley, G. G., Connolly, A., Weissman, I. L. and Longaker, M. T. (2016). Denervation of Mouse Lower Hind Limb by Sciatic and Femoral Nerve Transection. Bio-protocol 6(13): e1865. DOI: 10.21769/BioProtoc.1865.
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Category
Neuroscience > Neuroanatomy and circuitry > Sciatic nerve
Neuroscience > Neuroanatomy and circuitry > Femoral nerve
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1,866 | https://bio-protocol.org/exchange/protocoldetail?id=1866&type=0 | # Bio-Protocol Content
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A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii
Alberto J. Martín-Rodríguez
José J. Fernández
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1866 Views: 10696
Edited by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Jun 2015
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Jun 2015
Abstract
Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and Keshavan, 2006; Steindler and Venturi, 2007), or lethal gene fusions downstream QS-controlled promoters (Weiland-Bräuer et al., 2015). With three parallel QS circuits, the bioluminescent marine bacterium Vibrio campbellii (formerly harveyi, Lin et al., 2010) constitutes a complex Gram-negative model for which an extensive body of knowledge exists, including an array of mutant biosensors. In V. campbellii, bioluminescence is regulated by QS. However, bioluminescence is the result of complex biochemical networks that converge with cell respiration and fatty acid metabolism. It is also an energy-demanding reaction that strongly depends on the overall metabolic state of the bacterium, consuming up to 1/5 of the cell resources (Munn, 2011). Thus, disruption of QS-controlled phenotypes might be the result of toxic side effects or interference with the above-mentioned biochemical pathways rather than QS signaling. Therefore, adequate control experiments should be included. The protocol described herein provides a method and workflow for the identification of putative QS-disrupting compounds in Vibrio. It can also be easily adapted for other QS studies (e.g., detection of AI molecules).
Keywords: Quorum Sensing Vibrio Bioluminescence Inhibitors Screening
Materials and Reagents
White, clear-bottom 96-well plates (Sigma-Aldrich, Corning® Costar®, catalog number: 3610 )
Sterile sealing membrane (Sigma-Aldrich, Breathe-Easy®, catalog number: Z380059 )
15-ml tubes (VWR international, catalog number: 525-0150 )
96-well plate (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 167008 )
Cryovials (optional) (Cryoinstant) (Deltalab, catalog number: 409113/6 )
Disposable 1 mm path length cuvettes (Sigma-Aldrich, Brand®, catalog number: Z637092 )
Vibrio campbellii strain
Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
MgSO4 (Sigma-Aldrich, catalog number: M7506 )
Protein Hydrolysate Amicase (Sigma-Aldrich, catalog number: 82514 )
DI water
NaOH (Sigma-Aldrich, catalog number: S5881 )
Glycerol
Potassium phosphate monobasic (Sigma-Aldrich, catalog number: P5655 )
Potassium phosphate dibasic (Sigma-Aldrich, catalog number: 1551128 )
L-arginine (Sigma-Aldrich, catalog number: A5006 )
Kanamycin (Sigma-Aldrich, catalog number: K1377 )
Cinnamaldehyde (Sigma-Aldrich, catalog number: W228613 )
Marine Agar (MA) (Conda, catalog number: 1217 )
Autoinducer Bioassay (AB) medium (see Recipes)
Equipment
EnSpire® Multimode Plate Reader (PerkinElmer, model: 2300-0000 )
Tube rotator (Grant-bio, model: PTR35 )
Spectrophotometer (Beckman Coulter, model: DU 530 )
Software
GraphPad Prism (GraphPad Software, Inc.)
Microsoft Excel (Microsoft Corporation)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Martín-Rodríguez, A. J. and Fernández, J. J. (2016). A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii. Bio-protocol 6(14): e1866. DOI: 10.21769/BioProtoc.1866.
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Category
Microbiology > Microbial signaling > Quorum sensing
Microbiology > Microbial cell biology > Cell-based analysis
Biochemistry > Other compound > Flavonoid
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1,867 | https://bio-protocol.org/exchange/protocoldetail?id=1867&type=0 | # Bio-Protocol Content
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Isolation of Flavonoids from Piper delineatum Leaves by Chromatographic Techniques
Isabel L. Bazzocchi
Juan C. Ticona
Ignacio A. Jiménez
Ninoska Flores
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1867 Views: 17745
Edited by: Arsalan Daudi
Original Research Article:
The authors used this protocol in Jun 2015
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Jun 2015
Abstract
The genus Piper (Piperaceae) is widely distributed in the tropical and subtropical regions of the world, and species belonging to this genus are included in the Ayurvedic system of medicine and in folklore medicine of Latin America. Phytochemical investigations of Piper species have led to the isolation of several classes of physiologically active compounds such as alkaloids, amides, pyrones, dihydrochalcones, flavonoids, phenylpropanoids, lignans and neolignans. In an ongoing investigation of bioactive secondary metabolites from Piper species, herein, we describe the isolation procedure of nine flavonoids, including two chalcones and two flavanones from the leaves of Piper delineatum Trel. (Piperaceae), a shrub native to tropical regions of the Americas. All compounds were elucidated by spectroscopic and spectrometric methods, and comparison with data reported in the literature.
Keywords: Piper Flavonoids Isolation Chromatography
Materials and Reagents
Silica gel 60 (particle size 63-200 mm) (Macherey-Nagel, catalog number: 815330 )
Silica gel 60 (particle size 15-40 mm) (Macherey-Nagel, catalog number: 815650 )
TLC silica gel 60 F254 plates (Merck Millipore Corporation, catalog number: 105735 )
SIL G/UV254 (20 x 20 cm), Macherey-Nagel, catalog number: 805023 )
Pre-coated TLC-plates UV254 20 x 20 cm (Macherey-Nagel, catalog number: 821030 )
Round-bottom tubes (40 ml, 200 x 20 mm) (Simax, Alamo, catalog number: 00684800 )
Round-bottom tubes (8 ml, 120 x 12 mm) (Simax, Alamo, catalog number: 00684400 )
Microcapillary tube 1-5 μl (Sigma-Aldrich, catalog number: P0549-1PAK )
NMR simple tubes 5 mm (Wilmad® LabGlass, catalog number: 528-PP-7 )
Leaves of the plant species (Piper delineatum) at the mature stage were collected in Iquitos, Maynas Province, Department of Loreto, Perú in November 2009. A voucher specimen (10484) was identified by botanist Juan Ruiz Macedo and was deposited at the Amazonense Herbarium of Universidad Nacional de la Amazonia Peruana, Iquitos, Perú.
Ethanol absolute (Panreac, catalog number: 141086 )
Dichloromethane (CH2Cl) (Panreac, catalog number: 131254 )
Ethyl acetate (Panreac, catalog number: 141318.1212 )
Hexanes (Panreac, catalog number: 121347.1612 )
Chloroform (Panreac, catalog number: 161252 )
Diethyl ether (Panreac, catalog number: 142770.0311 )
Acetone (Panreac, catalog number: 131007.1212 )
Methanol (Panreac, catalog number: 141091.1211 )
Isopropanol (Panreac, catalog number: 131090 )
Acetone deuterated [(CD3)2CO] (Sigma-Aldrich, catalog number: 151793-25G )
Magnesium sulfate anhydrous (Panreac, catalog number: 212486 )
Sulfuric acid (Panreac, catalog number: 141058 )
Acetic acid glacial (Panreac, catalog number: 141008 )
Sea sand washed thin grain QP (Panreac, catalog number: 211160.0416 )
Sephadex LH-20 (Sigma-Aldrich, catalog number: LH20100 )
Oleum: Acetic acid-water-sulfuric acid (20:4:1) (see Recipes)
Coating rotor with sorbent for the CPTLC (4 mm thickness) (see Recipes)
Equipment
Rotary cutter mill (Mateu and Sole, S.L., Constructores, MATSO, model: B-2, nº 258 )
Soxhlet extraction apparatus (ANORSA, catalog number: 322060 )
Rotary R-210 evaporator (Sigma- Aldrich, Büchi® Rotavapor®, catalog number: Z565466 )
Separating funnel, 2 L (Sigma-Aldrich, catalog number: Z330663 )
Erlenmeyer flasks (100 ml and 500 ml) (Duran, catalog numbers: Z232793-1EA and Z232831-1EA , respectively)
Teflon funnel holder
Solvent-pouring funnel (stem L, O.D. 0.65 x 22 mm) (Sigma-Aldrich, catalog number: 548804 )
Evaporating flask (pear-shaped, 1L) (Sigma-Aldrich, catalog number: Z402990 )
Round-bottom flask (5 L) (Sigma-Aldrich, catalog number: Z302872 )
Glass Pasteur pipets 225 mm (BRAND, catalog number: 747720 )
Pasteur pipette rubber bulbs, 2 ml (Sigma-Aldrich, catalog number: Z111597-12EA )
Heating bath (Sigma-Aldrich, catalog number: Z563544 )
Heating mantle 5 L (Selecta, catalog number: 3031450 )
Hot plate (Selecta, catalog number: 1000443 )
Liebig Condenser (Sigma-Aldrich, catalog number: Z531006 )
Glass chromatography column (CC) for silica gel (50 x 9 cm) (Fisher Scientific, catalog number: 12058880 )
Glass chromatography column (CC) for Sephadex (60 x 4.5 cm) (Fisher Scientific, catalog number: 12011550 ).
Centrifugal preparative thin layer chromatography system (CPTLC) (Chromatotron, Harrison Research Inc., model: 7924T )
Silica gel PF254 disks (Merck Millipore Corporation, catalog number: 107749 2500 )
TLC Chamber rectangular for TLC plates (7.5 x 15.5 x 8.0 cm) (Sigma-Aldrich, catalog number: Z204226 )
TLC Chamber rectangular for PTLC plates (27.0 x 26.5 x 7.0 cm) (Sigma-Aldrich, catalog number: Z126195-1EA )
Glass atomizer reagent sprayer (125 ml) (Sigma-Aldrich, catalog number: Z529737-1EA )
Glass vacuum filter (Fisher Scientific, catalog number: 11979659 )
Micro-spatula (8 in) (Sigma-Aldrich, catalog number: Z513342-1PAK )
Analytical balance, semi-micro balance (Fisher Scientific, Mettler ToledoTM, catalog number: 11142062 ; model: NewClassic MS105DU)
Nuclear Magnetic Resonance (NMR) Spectrometers (Bruker, model: Bruker Avance 400 ; Bruker Avance 500 ).
Optical rotations polarimeter (CHCl3 at 25 °C) (PerkinElmer, model: 241 automatic polarimeter )
Ultraviolet (UV) spectrophotometer (JASCO, model: V-560 )
Infrared (IR) spectrophotometer (Bruker, model: IFS 55 , IFS 28/55 )
High resolution electron impact (HREI) mass spectrometer (MS) (MasSpec, model: Micromass VG Autospec magnetic sector , M series)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant biochemistry > Other compound
Plant Science > Plant metabolism > Other compound
Biochemistry > Other compound > Flavonoid
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1,868 | https://bio-protocol.org/exchange/protocoldetail?id=1868&type=0 | # Bio-Protocol Content
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Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum
Carmen Ruiz-Roldan
M. Isabel G. Roncero
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1868 Views: 10043
Edited by: Zhaohui Liu
Reviewed by: Shahin S. AliSusheel Kumar
Original Research Article:
The authors used this protocol in Jan 2015
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The authors used this protocol in:
Jan 2015
Abstract
To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.
Keywords: Glucose determination Fusarium Glucose utilization
Materials and Reagents
Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15 )
2 ml Eppendorf tubes
Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
5 mm-diameter glass beads (Sigma-Aldrich, catalog number: 18406-500G )
Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and Δcon7-1 mutant strains
Sterile dH2O
Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483 )
Glycerol (Merck, catalog number: 104092 )
Glucose (Coger SAS, catalog number: 24379.363 )
Na2CO3 (Sigma-Aldrich, catalog number: S2127 )
Glucose (GO) Assay Kit (Sigma-Aldrich, catalog number: GAGO-20 )
GO Assay includes:
Glucose Oxidase/Peroxidase Reagent (Sigma-Aldrich, catalog number: G3660 )
o-Dianisidine Reagent (Sigma-Aldrich, catalog number: D2679 )
Glucose Standard Solution (Sigma-Aldrich, catalog number: G3285 )
H2SO4, ACS reagent (Merck, catalog number: 108131.1000 )
MgSO4·7H2O (Merck, catalog number: 1058865.5000 )
KH2PO4 (Merck, catalog number: 104873.1000 )
KCl (Merck, catalog number: 104933.0500 )
NH4NO3 (Merck, catalog number: 101187.1000 )
FeSO4 (Merck, catalog number: 103965.0500 )
ZnSO4·7H2O (Merck, catalog number: 108883 )
MnSO4·H2O (Merck, catalog number: 105941 )
0.1% benzoic acid
Extraction buffer (see Recipes)
Synthetic medium (SM) (see Recipes)
H2SO4 (12 N) (see Recipes)
Equipment
Mini-BeadBeater -16 homogenizer (BioSpec Products)
Orbital incubator (Infors, Multitron Pro)
Sterile spatula (Fisher Scientific, catalog number: S50821 )
Microcentrifuge (Fisher Scientific, EppendorfTM MiniSpin plusTM)
Hemocytometer (Thoma) (Marienfeld, catalog number: 06 407 10 )
Fluorimeter (TECAN, SpectraFluorPlus, model: F129005 )
Freeze-dryer (Virtis, model: BT4KZL-105 )
Water bath (Selecta, model: 6000138 )
Vortex (IKA, model: MS2 Mini shaker )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ruiz-Roldan, C. and Roncero, M. I. G. (2016). Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum. Bio-protocol 6(14): e1868. DOI: 10.21769/BioProtoc.1868.
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Category
Biochemistry > Carbohydrate > Glucose
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1,869 | https://bio-protocol.org/exchange/protocoldetail?id=1869&type=0 | # Bio-Protocol Content
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Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum
Carmen Ruiz-Roldan
M. Isabel G. Roncero
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1869 Views: 9141
Edited by: Zhaohui Liu
Reviewed by: Shahin S. AliSusheel Kumar
Original Research Article:
The authors used this protocol in Jan 2015
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The authors used this protocol in:
Jan 2015
Abstract
Glycolysis provides metabolites for energy production via oxidative phosphorylation during vegetative growth of Fusarium oxysporum. Therefore, determination of intracellular ATP levels might be of valuable help to analyze regulation of glycolysis/gluconeogenesis pathways. The protocol described here can be applied to other filamentous fungi.
Keywords: ATP production Fusarium ATP quantification
Materials and Reagents
Monodur nylon filters 15 µm diameter (Filtravibracion S.L., Spain, catalog number: Nylon-15 )
Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
Eppendorf tube (2 ml)
5 mm-diameter glass beads (Sigma-Aldrich, USA, catalog number: 18406-500G )
Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and mutant strains.
Note: Strains used in this study are wild type strain 4287 (race 2) and Δcon7-1 and cΔcon7-1 mutant strains.
Glycerol (Merck, catalog number: 104092 )
Potato dextrose broth (PDB) (Scharlau, Spain, catalog number: 01483 )
Sterile dH2O
Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
EDTA (Amresco, catalog number: 0322 )
MgSO4·7H2O (Merck, catalog number: 1058865.5000 )
KH2PO4 (Merck, catalog number: 104873.1000 )
KCl (Merck, catalog number: 104933.0500 )
NH4NO3 (Merck, catalog number: 101187.1000 )
FeSO4 (Merck, catalog number: 103965.0500 )
ZnSO4·7H2O (Merck, catalog number: 108883 )
MnSO4 monohydrate (Merck, catalog number: 105941 )
Glucose (Coger SAS, catalog number: 24379.363 )
ATP determination kit (Thermo Fischer Scientific, Molecular Probes®, catalog number: A22066 ).
Kit contents:
D-Luciferin (Component A, MW 302, blue cap), 5 vials, each containing 3 mg of lyophilized powder
Luciferase, firefly recombinant (Component B, red cap) 40 μl of a 5 mg/ml solution in 25 mMTris-acetate, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v) glycerol and 30% (v/v) ethylene glycol
Dithiothreitol (DTT) (Component C, MW 154, black cap) 25 mg
Adenosine 5’-triphosphate (ATP) (Component D, Green cap), 400 μl of a 5 mM solution in TE buffer
20x Reaction Buffer (Component E) 10 ml of 500 mM
Tricine buffer, pH 7.8, 100 mM MgSO4, 2 mM EDTA and 2 mM sodium azide
Extraction buffer (see Recipes)
Potato dextrose broth (PDB) medium (see Recipes)
Equipment
Sterile spatula (Fisher Scientific, catalog number: S50821 )
Mini-BeadBeater-16 homogenizer (BioSpec Products)
Hemocytometer Thoma (Marienfeld, Germany, catalog number: 06 407 10 )
Orbital incubator (Infors, Multitron Pro, Switzerland)
Microcentrifuge (Eppendorf, MiniSpin plus) (Fisher Scientific)
Fluorimeter (TECAN, SpectraFluorPlus, catalog number: InfiniteM200PRO )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ruiz-Roldan, C. and Roncero, M. I. G. (2016). Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum. Bio-protocol 6(14): e1869. DOI: 10.21769/BioProtoc.1869.
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Category
Biochemistry > Other compound > Nucleoside triphosphate
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187 | https://bio-protocol.org/exchange/protocoldetail?id=187&type=0 | # Bio-Protocol Content
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Clonogenic Assay
XY Xiaodong Yang
Published: Vol 2, Iss 10, May 20, 2012
DOI: 10.21769/BioProtoc.187 Views: 97284
Original Research Article:
The authors used this protocol in Dec 2006
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Abstract
Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version (Franken et al., 2006).
Materials and Reagents
Cell culture medium
Phosphate buffered saline (PBS)
Fetal bovine serum (FBS)
Trypsin/ EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
Crystal violet (Sigma-Aldrich, catalog number: C3886 )
Methanol (Sigma-Aldrich, catalog number: 34860 )
Glacial acetic acid (Sigma-Aldrich, catalog number: 320099 )
Fixation solution
Colony fixation solution (see Recipes)
Crystal violet solution (see Recipes)
Equipment
Cell culture petri dishes or six-well plates (Thermo Fisher Scientific, catalog number: 08-772-1B )
Hemocytometer (Hausser Bright-Line) (Thermo Fisher Scientific, catalog number: 02-671-10 )
Stereomicroscope (e.g., Nikon Eclipse, model: TS100 )
Hemocytometer
Incubator
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yang, X. (2012). Clonogenic Assay. Bio-protocol 2(10): e187. DOI: 10.21769/BioProtoc.187.
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Category
Cancer Biology > Cell death > Cell biology assays
Cancer Biology > General technique > Drug discovery and analysis
Cell Biology > Cell viability > Cell death
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1,870 | https://bio-protocol.org/exchange/protocoldetail?id=1870&type=0 | # Bio-Protocol Content
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Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
Anthony Flamier
Andrea Barabino
Gilbert Bernier
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1870 Views: 9826
Reviewed by: Agnieszka PastulaNicoletta Cordani
Original Research Article:
The authors used this protocol in Oct 2015
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The authors used this protocol in:
Oct 2015
Abstract
Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.
Keywords: Cone photoreceptor Human Embryonic stem cell Default pathway Coco
Materials and Reagents
Tissue culture 6-well plates (Thermo Fischer Scientific, catalog number: 130184 )
Pipets (5 ml, 10 ml)
50 ml conical tubes (Thermo Fischer Scientific, catalog number: 339653CS )
Micro cover glass (VWR International, catalog number: 89015-724 )
Cell scraper (Thermo Fischer Scientific, catalog number: 179693 )
0.22 μm sterilize filter Filtropur (Sarstedt, catalog number: 83.1826.001 )
Human embryonic stem (hES) cell line HUES9 (Harvard stem cell institute) or H9 cells (WiCell, catalog number: WA09 )
hES cell media (Peprotech, catalog number: BM-HESC-500 )
N2 supplement (Gibco, catalog number: 17502-048 )
B27 supplement (Gibco, catalog number: 17504-044 )
DMEM/F12 (Gibco, catalog number: 11320-033 )
NEAA (Gibco, catalog number: 11140-050 )
Human FGF-basic (Peprotech, catalog number: 100-18B-50UG )
Heparin (Sigma-Aldrich, catalog number: H3149-10KU )
Human IGF-I (Peprotech, catalog number: 100-11 )
Human recombinant COCO (R&D Systems, catalog number: 3047-CC )
Accutase solution (100 ml) (Sigma-Aldrich, catalog number: A6964 )
Rock inhibitor Y-27632 (hydrochloride) (Cayman chemical, catalog number: 10005583 )
Reduced growth factor matrigel (Corning, catalog number: 354230 )
hES cell qualified matrigel (Corning, catalog number: 354277 )
Trizol reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596026 )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: 04693116001 )
Na2HPO4·7H2O (FisherBiotech, catalog number: BP332-500 )
NaCl (Amresco, catalog number: 0241 )
KCl (EMD Millipore, catalog number: PX1405-1 )
KH2PO4 (FisherBiotech, catalog number: BP362-1 )
Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
1x PBS
Antibodies against CRX, S-opsin, ABCA4 and cone arrestin
CI media (see Recipes)
10x phosphate-buffer saline (PBS) (see Recipes)
4% paraformaldehyde (PFA) (see Recipes)
hES-qualified matrigel plate coating (see Recipes)
Equipment
37 °C, 5% CO2 water jacketed incubator (Thermo Fisher Scientific, catalog number: 3110 )
P1000, P200 pipets
Microscope (Leica, model: DM IL )
Refrigerated Tabletop Centrifuge for 50 ml conical tubes (Thermo Fisher Scientific, Centra GP8R, 031220F )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Flamier, A., Barabino, A. and Bernier, G. (2016). Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors. Bio-protocol 6(14): e1870. DOI: 10.21769/BioProtoc.1870.
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Category
Developmental Biology > Cell growth and fate > Neuron
Stem Cell > Embryonic stem cell > Maintenance and differentiation
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1,871 | https://bio-protocol.org/exchange/protocoldetail?id=1871&type=0 | # Bio-Protocol Content
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Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection
Janis A. Müller
Jan Münch
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1871 Views: 9772
Edited by: Yannick Debing
Reviewed by: Vamseedhar RayaproluValeria Lulla
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear cells (PBMCs).
Keywords: HIV Semen Amyloid SEVI Sexual transmission
Materials and Reagents
Flat-bottom 96-well plates (Sarstedt AG, catalog number: 83.3924 )
White 96-well polystyrene plates (Thermo Fisher Scientific, NuncTM, catalog number: 136101 )
Round-bottom 96-well plates (Sarstedt AG, catalog number: 83.3925 )
V-bottom 96-well plates (Sarstedt AG, catalog number: 83.3926 )
Peripheral blood mononuclear cells (PBMCs)
Note: These cells were isolated from healthy blood donor buffy coats by Ficoll (Biocoll) gradient centrifugation (see: http://www.bio-protocol.org/e323), and stimulated with 1 µg/ml phytohaemagglutinin (PHA) and 10 ng/ml IL-2 for 3 days.
TZM-bl/JC53bl-13 (HeLa CD4+ CCR5+ LTR-luciferase and LTR-lacZ) cells (Wei et al., 2002) (National Institutes of Health AIDS Research and Reference Reagent Program, catalog number: 8129 )
β-Galactosidase Reporter Gene Assay System for mammalian cells (Thermo Fisher Scientific, Gal-ScreenTM, catalog number: T1027 )
Biocoll separating solution (Merck Millipore, Biochrom, catalog number: L6115 )
Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific, catalog number: 41965039 )
Luminescent Cell Viability Assay (Promega, CellTiter-Glo®, catalog number: G7571 )
Fetal bovine/calf serum (FCS) (Thermo Fisher Scientific, catalog number: 10270106 ) (inactivated for 30 min at 56 °C in a water bath)
Gentamicin (Thermo Fisher Scientific, catalog number: 15710049 )
HIV-1 stock generated by HEK293T cells transfected with proviral DNA (e.g., pBRHIV-1 NL4-3 or pBRHIV-1 NL4-3 92TH014-2, see Münch et al., 2007 and Kim et al., 2010) or from infected cells)
HIV-1_firefly-luciferase stock [e.g., NL4-3 HIV-1_Luciferase (e.g., similar to Hiebenthal-Millow and Kirchhoff, 2002)] generated by HEK293T cells transfected with a proviral DNA encoding firefly-luciferase
Human Interleukin-2 (IL-2) (Miltenyi Biotec, catalog number: 130-097-745 )
L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
Luciferase Assay System (Promega, catalog number: E1501 )
Penicillin-Streptomycin (PenStrep) (Thermo Fisher Scientific, catalog number: 15140122 )
Phosphate buffered saline (PBS), no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14190094 )
Phytohaemagglutinin (PHA) (Thermo Fisher Scientific, RemelTM, catalog number: R30852801 )
RPMI-1640 (Thermo Fisher Scientific, catalog number: 21875034 )
Semen (fresh or frozen)
Note: Ejaculates are collected from healthy individuals with informed consent. To minimize inter-donor variations, semen derived from > 10 individual donors are pooled and aliquoted. Before pooling (on ice) the ejaculates are allowed to liquefy for 20-30 min at room temperature. Pooled semen samples can be stored at -20 °C or -80 °C in 250 to 1,000 µl aliquots. In all experiments, semen aliquots need to be rapidly thawed, diluted, and mixed with virus. The remaining sample is discarded.
Complete medium for adherent TZM-bl cells (see Recipes)
Complete medium for suspension primary PBMCs (see Recipes)
Equipment
96-well plate centrifuge (Eppendorf, model: 5804 R ) equipped with A-2-MTP rotor
CO2 humidified incubator
5-50 µl 12-channel pipette (HTL, Discovery comfort, model: DV-12-50 )
20-200 µl 12-channel pipette (HTL, Discovery comfort, model: DV-12-200 )
Orion II Microplate Luminometer (Titertek Berthold, catalog number: 11300010 )
Software
Microsoft Excel (Microsoft)
Simplicity 4.02 (Berthold Detection Systems)
Procedure
Seed target cells
TZM-bl cells: Seed 10,000 TZM-bl cells per well in 96-well flat-bottom plates containing 100 µl complete medium.
PBMCs: Seed 200,000 stimulated PBMCs (1 µg/ml PHA + 10 ng/ml IL-2 for 3 days) per well in 96-well flat-bottom plates containing 100 µl complete medium.
Note: Fill the outer wells with 200 µl PBS to avoid evaporation of the inner wells containing the cells (Figure 1: wells in grey).
Culture 18-24 h at 37 °C in a 5% CO2 humidified incubator.
The next day, without removing the medium, add 180 µl fresh complete medium containing 100 µg/ml gentamicin (and 10 ng/ml IL-2 for PBMCs).
Note: Gentamicin prevents the outgrowth of bacteria that may have contaminated the semen collection container.
Dilute HIV-1 stock in the complete medium to achieve a MOI < 1 (e.g., 0.1, 0.01, 0.001).
Note: The HIV-1 enhancing activity of semen cannot be observed at high MOIs as all target cells have been already infected in the absence of semen.
Thaw semen samples quickly in a 37 °C water bath and immediately dilute in PBS to achieve semen concentrations of 0, 0.8, 4, 20, and 100% (v/v) in a 96-well round-bottom plate.
Note: Semen’s enhancing activity is lost over time. Avoid long room temperature exposure.
Add 40 µl of respective HIV-1 dilutions to 40 µl semen dilutions (1:1) in a 96-well round-bottom plate to achieve semen concentrations of 0, 0.4, 2, 10, and 50% (v/v).
Resuspend HIV-1/semen mixture; add 20 µl to 280 µl (1 to 15) cells in triplicates; the final cell culture concentrations of semen are 0, 0.027, 0.134, 0.67, 3.33% (v/v) (Figure 1).
Note: The low semen concentrations on the cells reduce its cytotoxic effect.
Incubate for 2-3 h at 37 °C in a 5% CO2 humidified incubator.
Thereafter, remove the medium and add 200 µl fresh complete medium containing 100 µg/ml gentamicin.
TZM-bl cells: Simple medium change is sufficient.
PBMCs: Centrifuge for 5 min at 300 x g at room temperature, discard 100 µl (of 300 µl) supernatant (without cells), resuspend cells and transfer the entire sample (200 µl) into a 96-well V-bottom plate. Centrifuge for 5 min at 300 x g at room temperature, discard supernatant, resuspend cells in 200 µl of fresh complete medium with 100 µg/ml gentamicin and 10 ng/ml IL-2 and transfer into 96-well F-bottom plates.
Note: Medium change prevents the toxic effects of semen.
Incubate at 37 °C in a 5% CO2 humidified incubator.
After 2 and 3 days, analyze all samples by light microscopy to detect the cytopathic effects (CPE) caused by HIV-1 infection, and the possible cytotoxic effects of semen.
Notes:
For TZM-bl cells: In this cell line, massive HIV-1 infection results in the formation of syncytia (see Figure 2 or supplementary Figure 1 in Kim et al., 2010). If syncytia are detectable at Day 2, determine the cellular β-galactosidase activities in all samples. Continued incubation of cultures that already show CPE for another 24 h may lead to over-infection and cell loss and should be avoided. If no CPE is detectable at Day 2, incubate cells for another 24 h, and determine reporter gene activities at Day 3 post infection. Final cell culture concentrations of semen of 3.33% are sometimes cytotoxic. Therefore, we greatly recommend running cytotoxicity assay in the absence of virus in parallel.
For PBMC: In PBMC, the HIV-1 induced CPE is less pronounced. If syncytia are detectable, determine the luciferase activities in all samples at Day 2. If no CPE is detectable at Day 2, incubate cells for another 24 h, and determine the reporter gene activities at Day 3 post infection. Note that PBMC are more susceptible to the cytotoxic effects of semen (Supplementary Figure 17 in Münch et al., 2007). Therefore, we greatly recommend running cytotoxicity assay in the absence of virus in parallel.
Determine reporter gene activities:
For TZM-bl cells (Figures 3 and 4) using the Gal-ScreenTM β-Galactosidase Reporter Gene Assay System
Remove supernatant.
Add 40 µl of lysis buffer together with substrate (mix according to the manufacturer’s instructions).
Incubate for 30 min at room temperature.
Transfer 35 µl of the lysed cells into a white 96-well luminometer plate.
Read luminescence as relative light units/s in a luminometer.
For PBMCs infected with an HIV-1 reporter virus encoding firefly luciferase using the Luciferase Assay System
Resuspend PBMCs and transfer into a 96-well V-bottom plate.
Centrifuge for 5 min, 550 x g at room temperature.
Discard supernatant.
Add 40 µl of 1x lysis buffer.
Incubate for 10 min at room temperature.
Resuspend and transfer 30 µl of the lysed sample into a white 96-well luminometer plate.
Add 100 µl substrate.
Immediately, read luminescence as relative light units/s in a luminometer.
Evaluate raw data (Figures 3 and 4).
Calculate average values obtained from non-infected cells (background) (Figure 3B).
Subtract average background values from each sample (Figure 3B).
Calculate average values and standard deviations of each triplicate measurement (Figures 3C and 4A).
Calculate n-fold enhancement values by setting reporter gene activities observed in the absence of semen = 1 (Figures 3D and 4B).
Representative data
Figure 1. Typical layout of a microtiter plate used in experiments to study the effect of semen on HIV-1 infection. Cells seeded in the inner 60 wells are inoculated with HIV-1 of indicated MOIs pretreated with 0, 0.4, 2, 10 and 50 % semen, or are left uninfected. grey: PBS; white: uninfected; colored: different MOIs of HIV-1.
Figure 2. Light microscopy analysis of infected TZM-bl cells in presence of semen. TZM-bl cells were inoculated with PBS (uninfected) or HIV-1 that has been preincubated with indicated concentrations of semen. Final semen concentrations on cells are given in brackets. Minor (0% semen), little (2% semen) and strong (10% semen) CPE. Scale bars are 100 µm.
Figure 3. Results and evaluation of a representative reporter gene assay. Different MOIs (red: 0.1, blue: 0.01, green: 0.001) of HIV-1 NL4-3 92Th014-2 (R5-tropic) were preincubated with indicated concentrations of semen before infection of TZM-bl cells. A. Raw data representing relative light units/second of β-galactosidase activity measured in each well. B. Average background (uninfected cells) is calculated and subtracted. C. Average and standard deviations of triplicate infections are calculated. D. Fold enhancement is calculated by setting 0% semen sample = 1.
Figure 4. Graphical presentation of data described above. A. Shown are average β-galactosidase activities (n = 3) measured 3 days after virus exposure. RLU/s: relative light units per sec. The numbers above the bars give n-fold enhancement of HIV infection by semen relative to that measured for the corresponding PBS control. B. n-fold enhancement values represented as bar graphs. Values represent average values obtained from triplicate infection ± standard deviation. Note that 50% semen during virion treatment (corresponding to final cell culture concentrations of semen of 3.3 %) are cytotoxic resulting in reduced infection rates.
Notes
The following issues need to be carefully considered:
Do not add DEAE-Dextran, polybrene, or other additives that are often used in TZM-bl assays to increase infection rates. Addition of separate enhancers will mask the infection enhancing activity of semen.
Use low MOIs that are either sub-infectious in the absence of semen or result in infection rates of less than 10%. Only then, an enhancing effect of semen on viral infection can be determined. Furthermore, a low MOI resembles physiological HIV-1 concentrations during sexual transmission.
The HIV-1 enhancing activity of semen decreases over time. Thus, semen needs to be handled quickly.
Pre-exposure of virus to semen allows binding of seminal amyloids to viral particles as it likely also occurs in vivo.
Semen, after collection, is often not sterile thus gentamicin needs to be added to avoid bacterial outgrowth.
Even low semen concentrations (starting from 1% on cells) are cytotoxic. Therefore, semen concentrations need to be reduced and the exposure time shortened (e.g., by media change).
In parallel, it is recommended to run cytotoxicity assays. [e.g., MTT assay (see Kim et al., 2010) or Luminescent Cell Viability Assay]
Assay does not need to be performed in flat 96-well plates, but can also be scaled into different formats.
If these points are considered, the experimental set up can be adapted and modified to allow measurement of infection of different target cells with any HIV strain and reporter virus (i.e., GFP followed by a flow cytometry readout), as well as other viruses such as HCMV and HSV (Tang et al., 2013; Torres et al., 2015).
Infection enhancement by in vitro generated amyloid fibrils can be examined using the same protocol, except that the toxicity avoiding steps, i.e., addition of gentamicin, high volumes of medium, removal of toxic semen after 2-3 h can be omitted.
Recipes
Complete medium for adherent TZM-bl cells
DMEM
10% heat-inactivated FCS
1% PenStrep
1% L-glutamine
(100 µg/ml gentamicin)
Complete medium for suspension primary PBMCs
RPMI
10% heat-inactivated FCS
1% PenStrep
1% L-glutamine
10 ng/ml IL-2
(100 µg/ml gentamicin)
Acknowledgments
This assay was first published in (Münch et al., 2007) and the protocol described in detail in (Kim et al., 2010). Thanks to Annika Röcker, Edina Lump, and Onofrio Zirafi for carefully reading and revising the protocol. Janis A. Müller is part of the International Graduate School in Molecular Medicine Ulm.
References
Arnold, F., Schnell, J., Zirafi, O., Sturzel, C., Meier, C., Weil, T., Standker, L., Forssmann, W. G., Roan, N. R., Greene, W. C., Kirchhoff, F. and Munch, J. (2012). Naturally occurring fragments from two distinct regions of the prostatic acid phosphatase form amyloidogenic enhancers of HIV infection. J Virol 86(2): 1244-1249.
Hiebenthal-Millow, K. and Kirchhoff, F. (2002) The most frequent naturally occurring length polymorphism in the HIV-1 LTR has little effect on proviral transcription and viral replication. Virology 292 (1), 169-75.
Kim, K. A., Yolamanova, M., Zirafi, O., Roan, N. R., Staendker, L., Forssmann, W. G., Burgener, A., Dejucq-Rainsford, N., Hahn, B. H., Shaw, G. M., Greene, W. C., Kirchhoff, F. and Munch, J. (2010). Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI. Retrovirology 7: 55.
Munch, J., Rucker, E., Standker, L., Adermann, K., Goffinet, C., Schindler, M., Wildum, S., Chinnadurai, R., Rajan, D., Specht, A., Gimenez-Gallego, G., Sanchez, P. C., Fowler, D. M., Koulov, A., Kelly, J. W., Mothes, W., Grivel, J. C., Margolis, L., Keppler, O. T., Forssmann, W. G. and Kirchhoff, F. (2007). Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131(6): 1059-1071.
Roan, N. R., Munch, J., Arhel, N., Mothes, W., Neidleman, J., Kobayashi, A., Smith-McCune, K., Kirchhoff, F. and Greene, W. C. (2009). The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection. J Virol 83(1): 73-80.
Roan, N. R., Muller, J. A., Liu, H., Chu, S., Arnold, F., Sturzel, C. M., Walther, P., Dong, M., Witkowska, H. E., Kirchhoff, F., Munch, J. and Greene, W. C. (2011). Peptides released by physiological cleavage of semen coagulum proteins form amyloids that enhance HIV infection. Cell Host Microbe 10(6): 541-550.
Roan, N. R., Liu, H., Usmani, S. M., Neidleman, J., Muller, J. A., Avila-Herrera, A., Gawanbacht, A., Zirafi, O., Chu, S., Dong, M., Kumar, S. T., Smith, J. F., Pollard, K. S., Fandrich, M., Kirchhoff, F., Munch, J., Witkowska, H. E. and Greene, W. C. (2014). Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection. J Virol 88(13): 7221-7234.
Tang, Q., Roan, N. R. and Yamamura, Y. (2013). Seminal plasma and semen amyloids enhance cytomegalovirus infection in cell culture. J Virol 87(23): 12583-12591.
Torres, L., Ortiz, T. and Tang, Q. (2015). Enhancement of herpes simplex virus (HSV) infection by seminal plasma and semen amyloids implicates a new target for the prevention of HSV infection. Viruses 7(4): 2057-2073.
Usmani, S. M., Zirafi, O., Muller, J. A., Sandi-Monroy, N. L., Yadav, J. K., Meier, C., Weil, T., Roan, N. R., Greene, W. C., Walther, P., Nilsson, K. P., Hammarstrom, P., Wetzel, R., Pilcher, C. D., Gagsteiger, F., Fandrich, M., Kirchhoff, F. and Munch, J. (2014). Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen. Nat Commun 5: 3508.
Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag, M. S., Wu, X., Shaw, G. M. and Kappes, J. C. (2002). Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46(6): 1896-1905.
Copyright: Müller and Münch. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Müller, J. A. and Münch, J. (2016). Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection . Bio-protocol 6(14): e1871. DOI: 10.21769/BioProtoc.1871.
Lump, E., Castellano, L. M., Meier, C., Seeliger, J., Erwin, N., Sperlich, B., Stürzel, C. M., Usmani, S., Hammond, R. M., von Einem, J., Gerold, G. 6., Kreppel, F., Bravo-Rodriguez, K., Pietschmann, T., Holmes, V. M., Palesch, D., Zirafi, O., Weissman, D., Sowislok, A., Wettig, B., Heid, C., Kirchhoff, F., Weil, T., Klärner, F. G., Schrader, T., Bitan, G., Sanchez-Garcia, E., Winter, R., Shorter, J. and Münch, J. (2015). A molecular tweezer antagonizes seminal amyloids and HIV infection. eLife 4: 05397.
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Category
Microbiology > Microbe-host interactions > Virus
Microbiology > Microbial cell biology > Cell-based analysis
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1,872 | https://bio-protocol.org/exchange/protocoldetail?id=1872&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)
Janna M. Bigalke
Ekaterina E. Heldwein
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1872 Views: 7814
Edited by: Arsalan Daudi
Reviewed by: Balasubramanian Venkatakrishnan
Original Research Article:
The authors used this protocol in Dec 2015
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Original research article
The authors used this protocol in:
Dec 2015
Abstract
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.
Keywords: Nuclear egress complex Protein expression in E. coli Protein purification Crystallization
Materials and Reagents
Talon HiTrap columns, 1 ml (GE Healthcare, catalog number: 28-9537-66 )
24-well crystallization plates, VDX Greased Plates (Hampton Research Corporation, catalog number: HR3-170 )
Cover slips, 22 mm, siliconized (Hampton Research Corporation, catalog number: HR3-233 )
Nylon hydrophilic membrane filters, 0.2 µm (Merck Millipore Corporation, catalog number: GNWP04700 )
Ultrafree centrifugal filters, 0.1 µm (Merck Millipore Corporation, catalog number: UFC30VV00 )
LB (Luria Bertani) agar plates
Escherichia coli (E. coli) BL21 Rosetta (DE3) (Merck Millipore Corporation, Novagen, catalog number: 70954 )
pGEX-6P1 vector (GE Healthcare, catalog number: 28-9546-48 )
pET-24b vector (Merck Millipore Corporation, catalog number: 69750-3 ), modified to include a sequence encoding a His6-SUMO tag followed by a PreScission cleavage site
Ampicillin (Fisher Scientific, catalog number: BP1760-25 )
Kanamycin (Fisher Scientific, Durable, catalog number: BP906-5 )
Chloramphenicol (Fisher Scientific, Bioreagents, catalog number: BP904-100 )
Tryptone (Fisher Scientific, catalog number: BP1421-2 )
Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
Glycerol (P212121, catalog number: RP-G22020-0.5 )
KH2PO4 (Fisher Scientific, catalog number: P386-500 )
K2HPO4 (Fisher Scientific, catalog number: LC200901 )
Glucose (American Bio, catalog number: AB00715 )
Lactose (Fisher Scientific, Durable, catalog number: L5-500 )
MgSO4·7H2O (Sigma-Aldrich, Fluka, catalog number: 63140 )
EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich, cOmpleteTM, catalog number: 5056489001 )
Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25-1 g )
HEPES (Fisher Scientific, Durable, catalog number: BP310500 )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Durable, catalog number: S271-10 )
TCEP HCl (P212121, catalog number: SV-TCEP-25 g )
Imidazole (Thermo Fisher Scientific, ACROS Organics, catalog number: 301870010 )
Ni Sepharose 6 Fast Flow (GE Healthcare, catalog number: 17-5318-02 )
Glutathione Sepharose 4B (GE Healthcare, catalog number: 17-0756-05 )
PreScission protease (produced in house; for more information see Reference 3)
12% Mini Protean TGX precast gels (Bio-Rad Laboratories, catalog number: 456-1046 )
Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, catalog number: 161-0400 )
Acetic acid (Fisher Scientific, Durable, catalog number: A491-212 )
Ethanol, 190 proof (Fisher Scientific, Durable, catalog number: 04-355-226 )
Guanidine hydrochloride (Sigma-Aldrich, catalog number: 50950 )
Trisodium citrate dihydrate (Alfa Aesar, catalog number: 36439-A3 )
NiCl2 hexahydrate (Alfa Aesar, catalog number: 43185 )
Polyethylene glycol 8000 (Sigma-Aldrich, catalog number: 89510 )
NaSCN (VWR International, catalog number: 10118-142 )
Polyethylene glycol 3350 (Sigma-Aldrich, catalog number: P3640 )
Note: Product P3640 has been discontinued.
Meso-erythritol (VWR International, catalog number: CAAAA15813-22 )
TB (Terrific broth) medium (see Recipes)
Lysis buffer (see Recipes)
Gel filtration buffer (see Recipes)
Equipment
Standard laboratory equipment
Spectrophotometer device (Thermo Fisher Scientific, NanoDropTM, model: 1000 ) or comparable UV/VIS spectrophotometer
Note: This product has been discontinued by the manufacturer.
Microfluidizer cell disruptor (Microfluidics, model: 110S ) or comparable cell lysis equipment
Note: This product has been discontinued by the manufacturer.
Pumps (Bio-Rad Laboratories, model: EP-1 )
Note: This product has been discontinued by the manufacturer.
Chromatography system (Bio-Rad Laboratories, DuoFlowTM, model: Medium-pressure ) or comparable chromatography system
Superdex 75 10/300 (GE Healthcare, catalog number: 17-5174-01 )
Stereo microscope (ZEISS, model: SteREO Discovery V8 ) or comparable microscope
Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane (Merck Millipore Corporation, catalog number: UFC905096 )
Ultrasonic bath (Fisher Scientific, model: FS60) or comparable sonication device
Note: This product has been discontinued by the manufacturer.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Bigalke, J. M. and Heldwein, E. E. (2016). Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC) . Bio-protocol 6(14): e1872. DOI: 10.21769/BioProtoc.1872.
Bigalke, J. M. and Heldwein, E. E. (2015). Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J 34(23): 2921-2936.
Download Citation in RIS Format
Category
Biochemistry > Protein > Isolation and purification
Biochemistry > Protein > Expression
Biochemistry > Protein > Structure
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1,873 | https://bio-protocol.org/exchange/protocoldetail?id=1873&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
JP Jeannette Pfalz
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1873 Views: 8700
Edited by: Marisa Rosa
Original Research Article:
The authors used this protocol in May 2015
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The authors used this protocol in:
May 2015
Abstract
The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
Keywords: Enrichment of the plastid encoded Polymerase (PEP) RNA Polymerase assembly Blue native PAGE (BN-PAGE) Sucrose gradient centrifugation Plastid protein complexes
Materials and Reagents
Material
Disposable RNase-free pipette tips (1-200 µl and 100-1,000 µl universal tips)
RNase-free microcentrifuge tubes (1.5 ml and 2.0 ml universal tubes)
15 ml conical tube
Amicon Ultra-15 centrifugal filter units, 10KDa (Merck Millipore Corporation, catalog number: UFC901024 )
PVDF-Membrane (Carl Roth, Roti® -Fluoro, catalog number: 2803.1 )
Polypropylene centrifuge tubes, 14 x 95 mm (Beckman, catalog number: 331374 )
Plant material
Maize seedlings grown in soil for 7-10 days at 26-28 °C in cycles of 16 h-light/8 h-dark
Antibodies
anti-ZmpTAC12 (custom antibody) (Biogenes)
anti-ZmRpoA (custom antibody) (Biogenes)
Goat anti-Rabbit IgG HRP-linked (Sigma-Aldrich, catalog number: A6154 )
Reagent
Liquid nitrogen
Acrylamide, Gel 40 (29:1) (Carl Roth, Rotiphorese®, catalog number: A515.1 )
Ammonium persulfate (APS), (NH4)2S2O8 (Sigma-Aldrich, catalog number: 248614-500G-D )
Bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane, Bis-Tris (Carl Roth, catalog number: 9140.3 )
β-Mercaptoethanol (Carl Roth, catalog number: 4227.1 )
Bradford, Roti®-Nanoquant (Carl Roth, catalog number: K880.1 )
Coomassie brilliant blue G250 (Sigma-Aldrich, catalog number: B0770-5G )
1, 4-dithiothreitol, DTT (Carl Roth, catalog number: 6908.1 )
ε-aminocaproic acid (Carl Roth, catalog number: 3113.2 )
Glycerol (Carl Roth, catalog number: 6962.3 )
Glycine (Carl Roth, catalog number: 3187.4 )
HEPES (Carl Roth, catalog number: 9105.3 )
Magnesium acetate tetrahydrate, Mg(CH3COO)2·4H2O, (Carl Roth, catalog number: 0 275.2 )
Magnesium chloride, MgCl2 (Carl Roth, catalog number: KK36.3 )
Methanol (Carl Roth, catalog number: P717.1 )
n-Dodecyl β-D-maltoside, β-DM (Sigma-Aldrich, catalog number: D4641-1G )
N, N, N’, N’-Tetramethylethylenediamine (TEMED) (Carl Roth, catalog number: 2367.1 )
N-Tris-(hydroxymethyl)-methyl-glycin, Tricin (Carl Roth, catalog number: 6977.3 )
BlueEasy Prestained Protein Marker (Nippon Genetics, catalog number: MWP06 )
Potassium acetate, K(CH3COO) (Carl Roth, catalog number: T874.1 )
Potassium hydroxide (Carl Roth, catalog number: 7986.1 )
Protease inhibitor cocktail (cOmplete)
Note: Currently, it is “Sigma-Aldrich, cOmpleteTM, catalog number: 000000011836153001 ”.
Tris-(hydroxymethyl)-aminomethan, TRIS (Carl Roth, catalog number: 4855.3 )
Triton X-100 (Carl Roth, catalog number: 3051.2 )
Tween-20 (Carl Roth, catalog number: 9127.1 )
Sodium chloride, NaCl (Carl Roth, catalog number: 9265.2 )
Sodium dodecyl sulfate, SDS (Carl Roth, catalog number: 4360.2 )
Sodium fluorid, NaF (Carl Roth, catalog number: 4503.2 )
D-sucrose [D(+)-saccharose] Carl Roth, catalog number: 4621.2 )
Buffers
BN-Lysis buffer (see Recipes)
3x BN-Gel buffer (see Recipes)
4.5% Separating gel (see Recipes)
14% Separating gel (see Recipes)
4% Stacking gel (see Recipes)
Cathode buffer blue (see Recipes)
Cathode buffer (see Recipes)
Anode buffer (see Recipes)
2x BisTris ACA (ACA, aminocaproic acid) (see Recipes)
Sample buffer (see Recipes)
2% n-dodecyl-β-D-maltopyranoside (β-DM) solution (see Recipes)
BN-Loading buffer (see Recipes)
Transfer buffer (see Recipes)
1x TBST buffer (see Recipes)
Low sucrose solution (see Recipes)
High sucrose solution (see Recipes)
Diluent solution (see Recipes)
Equipment
Mortar and pestle
Sonicator (BANDELIN electronic GmbH & Co., model: Sonopuls HD 2200 )
Microcentrifuge (Beckman, model: Avanti JXN-30 )
Ultracentrifuge (Sorvall Discovery 90SE)
Note: This product has been discontinued by the manufacturer.
SW40 rotor (Beckman)
Gradient mixer (BIO-RAD, model: 385 )
Note: This product has been discontinued by the manufacturer.
BioPhotometer (Eppendorf)
Electrophoresis apparatus (Hoefer, model: SE600 )
Semi-Dry-Blotter (VWR International, PEQLAB Biotechnologie GmbH)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pfalz, J. (2016). Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation . Bio-protocol 6(14): e1873. DOI: 10.21769/BioProtoc.1873.
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Category
Biochemistry > Protein > Isolation and purification
Plant Science > Plant biochemistry > Protein
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1,874 | https://bio-protocol.org/exchange/protocoldetail?id=1874&type=0 | # Bio-Protocol Content
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Peer-reviewed
Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
David Canadell
Samuel Bru
Josep Clotet
Joaquín Ariño
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1874 Views: 11471
Edited by: Fanglian He
Reviewed by: Yanjie LiRosario Gomez-Garcia
Original Research Article:
The authors used this protocol in Feb 2015
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The authors used this protocol in:
Feb 2015
Abstract
Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the polyP values obtained.
Figure 1. Enzymatic hydrolysis of polyP
Keywords: Yeast Polyphosphate Extraction methods Enzymatic phosphate release
Materials and Reagents
1.5 ml screw cap tubes
Silica-gel columns from QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
Inoculation loop
Magnetic stirrer
96-Well assay microplate non-treated clear polystyrene (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 269620 )
Yeast Saccharomyces cerevisiae
Yeast extract, peptone (YP) base medium (Conda, catalog number: 1511 )
AE buffer
Phenol solution (Sigma-Aldrich, catalog number: P4557 )
Sodium dodecylsulfate (SDS) (Panreac AppliChem, catalog number: A7249 )
Chloroform (CHCl3) (Merck Millipore, catalog number: 102445 )
RNase A (100 mg/ml) (QIAGEN, catalog number: 19101 )
DNase I (Roche Diagnostics, catalog number: 10104159001 )
Sodium acetate trihydrate (NaC2H3O2·3H2O) (Merck Millipore, catalog number: 106267 )
Ethanol absolute (CH3CH2OH) (Panreac Applichem, catalog number: 131086 )
Milli-Q water
98% sulfuric acid (H2SO4) (Merck Millipore, catalog number: 112080 )
Sodium hydroxide (NaOH) (Panreac Applichem, catalog number: 131687 )
Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer (Panreac Applichem, catalog number: A1379 )
Neutral red (Sigma-Aldrich, catalog number: N4638 )
Sodium iodide (NaI) (Sigma-Aldrich, catalog number: 383112 )
Acetic acid glacial (CH3COOH) (Panreac Applichem, catalog number: 131088 )
Disodium ethylenediaminetetraacetate 2-hydrate (EDTA) (Panreac Applichem, catalog number: 131669 )
Recombinant Ppx1 (rPpx1)
Note: Ppx1 is a S. cerevisiae exopolyphosphatase that hydrolyses polyphosphate into Pi residues. It was obtained from E. coli containing a plasmid-borne, His-tagged version of the PPX1 gene, as described in (Ruiz et al., 2001), after one-step affinity purification with HisTrapTM HP columns (GE Healthcare Life Sciences ).
Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore, catalog number: 104877 )
Sodium phosphate glass Type 45 (polyP45) (Sigma-Aldrich, catalog number: S4379 )
Sodium tripolyphosphate (polyP3) (Sigma-Aldrich, catalog number: 238503 )
Malachite green oxalate salt (Sigma-Aldrich, catalog number: M9015 )
Polyvinyl alcohol (Sigma-Aldrich, catalog number: P1763 )
D-Glucose monohydrate (Panreac Applichem, catalog number: A1349 )
Ammonium acetate (NH4C2H3O2) (Panreac Applichem, catalog number: 131114 )
Magnesium acetate tetrahydrate [Mg(CH3COO)2·4H2O] (Merck Millipore, catalog number: 105819 )
Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sigma-Aldrich, catalog number: 09878 )
YPD (see Recipes)
3 M sodium acetate (see Recipes)
0.5 M EDTA (see Recipes)
Buffer AE (see Recipes)
10% SDS (see Recipes)
RNase A (10 mg/ml) (see Recipes)
DNase I (10 mg/ml) (see Recipes)
70% ethanol (see Recipes)
0.1% neutral red solution (see Recipes)
1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v) (see Recipes)
2 M NaOH (see Recipes)
1 M sulfuric acid (see Recipes)
6 M NaI (see Recipes)
1 M ammonium acetate (see Recipes)
1 M magnesium acetate (see Recipes)
5 M NaCl (see Recipes)
Wash buffer (see Recipes)
Polyphosphate assay buffer (see Recipes)
50 mM potassium dihydrogen phosphate (see Recipes)
Phosphate calibration curve (see Recipes)
222 µM polyP45 (10 mM Pi) (see Recipes)
3.3 mM polyP3 (10 mM Pi) (see Recipes)
6 µM polyP45 (see Recipes)
200 µM polyP3 (see Recipes)
28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes)
0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes)
Equipment
Novaspec Plus spectrophotometer GE
Thermo LabSystems Multiskan Ascents 354 microplate reader
Eppendorf Thermomixer® compact
Vortex mixer (Heidolph)
Centrifuge (Eppendorf, MiniSpin®)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Canadell, D., Bru, S., Clotet, J. and Ariño, J. (2016). Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae. Bio-protocol 6(14): e1874. DOI: 10.21769/BioProtoc.1874.
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Category
Microbiology > Microbial biochemistry > Other compound
Microbiology > Microbial biochemistry > Protein
Biochemistry > Other compound > Ion
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1,875 | https://bio-protocol.org/exchange/protocoldetail?id=1875&type=0 | # Bio-Protocol Content
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Peer-reviewed
House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis
Chris Tibbitt
Jonathan M. Coquet
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1875 Views: 12107
Edited by: Ivan Zanoni
Reviewed by: Meenal SinhaMario M. Zaiss
Original Research Article:
The authors used this protocol in Aug 2015
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The authors used this protocol in:
Aug 2015
Abstract
Asthma is a complex disease of the airways primarily mediated by T helper 2 cells and innate lymphoid type 2 cells (Licona et al.,2013). Mice do not develop spontaneous asthma and therefore models have been developed for the assessment of key processes that underlie human pathology (Nial et al.,2008). Exposure to House Dust Mite (HDM) extract induces many key features of acute airway inflammation including elevated IgE levels, eosinophilia, goblet cell metaplasia, epithelial hypertrophy and airway hyperresponsiveness (AHR) in response to methacholine (Hammad et al., 2009; Dullaers et al., 2012; Coquet et al., 2015). The exact dose and duration of exposure to HDM can affect the type and extent of inflammation. In our case, we start with a low sensitizing dose that is increased on challenge, while others use differing schedules or a higher antigen concentration during sensitization of mice (Hondowicz et al., 2016; Trompette et al., 2014; Zaiss et al., 2015). We believe that using a low sensitizing dose more accurately separates the primary and secondary immune responses and reduces the possibility that HDM given during sensitization continues to fuel the immune response during challenge (Coquet et al., 2015; Plantinga et al., 2013). Here, we outline in text, pictures and video how to administer HDM extracts or cytokines via the intranasal route and briefly touch upon the subsequent analysis of inflammation in the airways [covered otherwise in ( Han et al., 2013)].
Keywords: HDM IL-33 Asthma Lavage Airway Inflammation
Materials and Reagents
1.5 ml Eppendorf tube (Sarstedt, catalog number: 72.269.001 )
100 μM Filter (BD, catalog number: 352360 )
Note: Currently, it is “Corning, FalconTM, catalog number: 352360”.
Sterile pipette tips (Corning, catalog number: 4804 )
23 Gauge (G) sterile needle (BD, catalog number: 300800 )
Polyethylene tubing PE-10 (BD, IntramedicTM, catalog number: 427401 )
1 ml syringe (BD, catalog number: 309659 )
15 ml screw cap tube conical (Corning, FalconTM, catalog number: 352096 )
50 ml screw cap tube conical (Corning, FalconTM, catalog number: 352070 )
Soft wood/styrofoam board
Mice
Note: We typically utilize C57BL/6 mice of at least 4 weeks of age. Either sex can be used although it is best not to mix genders in a particular experiment.
Isoflurane (Baxter, catalog number: KDG9623 )
House Dust Mite (HDM) extract (Greer Laboratories, catalog number: XPB70D3A2.5 )
1x Dulbecco’s phosphate buffered saline (DPBS) (Sigma-Aldrich, catalog number: D8537 )
Ethanol
Recombinant cytokines [e.g., rIL-21 (gifted by Zymogenetics); rIL-25 (Biolegend, catalog number: 587302 ) or IL-33 (Biolegend, catalog number: 580502 )]
Fetal calf serum (FCS), heat inactivated (Sigma-Aldrich, catalog number: F4135 )
Trypan blue (Sigma-Aldrich, catalog number: 93595 )
Red blood cell lysis buffer (Sigma-Aldrich, catalog number: R7757 )
Equipment
Laminar flow hood
Rodent anesthesia system [e.g., Univentor 400 Anesthesia Unit or Univentor 410 Anesthesia unit (Univentor, catalog number: 8323101 )]
Pipette p200 (Sigma-Aldrich, catalog number: Z678333 )
Spray bottle with 70% ethanol (Sigma-Aldrich, catalog number: Z560847 )
Surgical scissors (e.g., Sigma-Aldrich, catalog number: Z265969 )
Surgical Thread 4-0 (0.17 mm), sterile (e.g., Agnthos, catalog number: 14757 )
Stainless steel forceps (e.g., Sigma-Aldrich, catalog number: Z168785 )
Hemocytometer
Centrifuge [e.g., Hettich Rotina 420R (Sigma-Aldrich, catalog number: Z723630 )]
Figure 1. Anesthesia setup. A. Mice should be anesthetized by isoflurane with the rate of breathing carefully observed to ensure a sufficient degree of anesthesia without being too light or too deep. B. Example of anesthesia system in this case is the Univentor 400 with air flow set to 388 ml/min and Isoflurane concentration of 3.8%.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tibbitt, C. and Coquet, J. M. (2016). House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis. Bio-protocol 6(14): e1875. DOI: 10.21769/BioProtoc.1875.
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Category
Immunology > Animal model > Mouse
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1,876 | https://bio-protocol.org/exchange/protocoldetail?id=1876&type=0 | # Bio-Protocol Content
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Peer-reviewed
Mouse Subependymal Zone Explants Cultured on Primary Astrocytes
Ionut Dumitru
HM Hannah Monyer
Julieta Alfonso
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1876 Views: 7742
Edited by: Oneil G. Bhalala
Reviewed by: Joseph C. Chen
Original Research Article:
The authors used this protocol in Oct 2015
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The authors used this protocol in:
Oct 2015
Abstract
Neuroblast migration is a major component in the process of neuronal circuit assembly. In the rodent postnatal brain, the subependymal zone (SEZ) is the largest neurogenic niche where new neurons are born throughout life. These cells migrate several millimeters following a specific pathway called the rostral migratory stream (RMS) towards the olfactory bulb. Once they reach their final destination, they differentiate mainly as interneurons and integrate into already established neuronal circuits. Along the RMS, neuroblasts use a particular mode of migration known as chain migration. They stay attached to each other while migrating and are intimately associated with adjacent astrocytic processes. To dissect the molecular and cellular signals involved in neuroblast migration, we developed an in vitro system consisting of SEZ-derived explants co-cultured on top of an astrocyte monolayer.
Keywords: Neuroblast Neurogenesis Migration RMS Postnatal
Materials and Reagents
10 cm Petri dishes (Greiner Bio-One GmbH, catalog number: 664160 )
15-ml tube
75 cm2 plastic flasks (Sigma-Aldrich, Corning® CellBIND®, catalog number: CLS3290 )
Glass coverslips (Marienfeld-superior 18 mm Φ)
24-well plates (Sigma-Aldrich, Greiner Cellstar®, catalog number: M8812-100EA )
Breakable razor blades (Fine Science Tools, catalog number: 10050-00 )
Puradisc 30 syringe filters (Whatman, Whatman®, catalog number: 10462200 )
Animals: Wild-type C57BL/6 mice (Charles River)
Note: Animal care and procedures were according to local and international regulations for the use of experimental animals. Animal handling was performed by trained staff certified by the Federation of European Laboratory Animal Science Associations (FELASA).
Antibodies:
Goat anti-DCX (Santa Cruz Biotechnology, catalog number: sc-8066 )
Mouse anti-GFAP (Sigma-Aldrich, catalog number: G-3893 )
Anti-goat conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A11055 )
Anti-mouse conjugated to Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A31571 )
Papain (Sigma-Aldrich, catalog number: P4762 )
DNase I (Sigma-Aldrich, catalog number: D4513 )
Dulbecco’s modified Eagle’s media (DMEM) (Thermo Fisher Scientific, catalog number: 41965039 )
Fetal bovine serum (FBS), heat inactivated (Thermo Fisher Scientific, catalog number: 10270-106 )
Trypsin–EDTA (0.05%) (Thermo Fisher Scientific, catalog number: 25300-054 )
Poly-L-lysine (Sigma-Aldrich, catalog number: P9155 )
Neurobasal media (Thermo Fisher Scientific, 21103049 )
4% paraformaldehyde (PFA) (Carl Roth, catalog number: P087.3 )
Phosphate buffer saline (PBS)
PBS-Triton
Bovine Serum Albumin (BSA)
Mowiol (Sigma-Aldrich, catalog number: 324590 )
NaCl (Sigma-Aldrich, catalog number: 31434-1KG-R )
KCl (AppliChem GmbH, catalog number: A1039 )
Na2HPO4 (VWR International, Prolabo, catalog number: 28029.292 )
KH2PO4 (GERBU Biotechnik, catalog number: 2018 )
Glucose (Sigma-Aldrich, catalog number: G-7021 )
HEPES (Carl Roth Gmbh, catalog number: 9105.2 )
B27 supplement (Thermo Fisher Scientific, 17504044 )
L-glutamine (Thermo Fisher Scientific, catalog number: 25030-024 )
Pen/Strep (Thermo Fisher Scientific, catalog number: 15140-122 )
PBS-Glucose-HEPES (see Recipes)
Neurobasal complete media (see Recipes)
Dulbecco’s modified Eagle’s medium (DMEM)-10% serum (see Recipes)
Papain solution (see Recipes)
DNase I solution (see Recipes)
Equipment
Standard pattern scissors sharp/blunt 14,5cm (Fine Science Tools, catalog number: 14101-14 )
Stereomicroscope (Carl Zeiss, model: STEMI SV 6 ) supplemented with a halogen light source (Schott KL, model: 1500 LCD )
Horizontal laminar flow bench (Azbil Telstar, model: H-100 )
Biological safety cabinet (The Baker Company, model: SterilGARD e3 )
Dumont #5 Forceps (Fine Science Tools, catalog number: 11251-20 )
Graefe forceps-straight/serrated (Fine Science Tools, catalog number: 11050-10 )
Neubauer chamber
Scalpel Handle #3-12 cm (Fine Science Tools, catalog number: 10003-12 )
Scalpel Blades #15 (Fine Science Tools, catalog number: 10015-00 )
37 °C, 5% CO2 incubator (Labotect, model: C200 )
Bright field microscope
Confocal microscope (Carl Zeiss, model: LSM 700 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Dumitru, I., Monyer, H. and Alfonso, J. (2016). Mouse Subependymal Zone Explants Cultured on Primary Astrocytes . Bio-protocol 6(14): e1876. DOI: 10.21769/BioProtoc.1876.
Alfonso, J., Penkert, H., Duman, C., Zuccotti, A. and Monyer, H. (2015). Downregulation of Sphingosine 1-Phosphate Receptor 1 Promotes the switch from tangential to radial migration in the OB. Journal of Neuroscience 35(40): 13659-13672.
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell isolation and culture > Cell isolation
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1,877 | https://bio-protocol.org/exchange/protocoldetail?id=1877&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues
Vamsi J Nalam
Sujon Sarowar
Jyoti Shah
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1877 Views: 12490
Edited by: Arsalan Daudi
Reviewed by: Sara Posé Shweta Kalve
Original Research Article:
The authors used this protocol in Oct 2015
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Oct 2015
Abstract
Fusarium graminearum (Fg) is the causal agent of Fusarium head blight disease of wheat (Triticum aestivum), oats (Avena sativa) and barley (Hordeum vulgare), which targets the floral tissues and thereby adversely impacts grain yield and quality. Mycotoxins produced by F. graminearum further limit the consumability of infected grain. In the laboratory, F. graminearum also has the ability to colonize both leaves and inflorescence tissues of Arabidopsis thaliana. The interaction between A. thaliana and F. graminearum makes available a large array of genetic and molecular tools to study the interaction between plants and F. graminearum to elucidate plant genes and pathways that contribute to resistance, as well as study how the fungus targets plant genes and mechanisms to promote disease. The methods described below allow for efficient infection of Arabidopsis leaves and inflorescence, and evaluation of disease progress and fungal growth. Disease spread in Arabidopsis can be readily monitored by the visual observations of chlorosis of leaf tissue and disease phenotype of inflorescence tissue including fungal mass on surface of the inflorescence tissue. Fungal growth can be further monitored by measuring the relative amount of Fg DNA in the host tissue by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR).
Materials and Reagents
PCR tubes (Fisher Scientific, catalog number: 14222 262 )
Petri dishes (100 x 15 mm) (Fisher Scientific, catalog number: FB0875713 )
50 ml plastic screw-capped tubes (Midsci, catalog number: C50B )
Pipette tips (sterile) (Midsci, catalog number: AVR-1, AVR-4 and AVR-11 )
1.7 ml microfuge tubes (sterile) (Catalog number: AVSS1700 )
Cheesecloth from a local craft store or Miracloth (EMD Millipore, catalog number: 475855-1R )
Culture tube
1 ml needle-less syringe (Tuberculin syringe) (Becton Dickinson, catalog number: 309659 )
Funnel
1 L glass conical flask (Pyrex brand)
Tweezers
Hemocytometer
Camel hair brush
Sharpie or comparable water-proof marker
Disposable gloves
Kimwipes, tissue paper or paper towels
Face shield (Fisher Scientific, catalog number: 18-999-4542 )
Kord brand 3.5 inch square pots with bottom holes (Hummert International, catalog number: 12-1350-1 )
T.O. Plastics Standard Flats 1020 tray with bottom holes (Hummert International, catalog number: 11-3000-1 )
T.O. Plastics Standard Flats 1020 tray without holes (Hummert International, catalog number: 11-3050-1 )
DOM1020 plastic dome to fit 1020 flats (Hummert International, catalog number: 11-3360-1 )
Transparent plastic bags (Glad 13 gallon Recycling Drawstring Clear Trash bag)
Fusarium graminearum isolate Z-3639 (Bowden and Leslie, 1999)
Arabidopsis thaliana seeds (Accession Columbia, Nössen, and Wassilewskija)
Silwet L-77 (Lehle seeds, catalog number: VIS-30 )
Potato Dextrose Broth (Becton Dickinson, catalog number: 254920 )
Yeast extract (Becton Dickinson, catalog number: 212750 )
BD Difco Agar (Becton Dickinson, catalog number: 214530 )
Ammonium Nitrate (Fisher Scientific, catalog number: A676 )
Potassium chloride (Fisher Scientific, catalog number: P217 )
Magnesium sulfate heptahydrate (Fisher Scientific, catalog number: M63 )
Sodium chloride (Fisher Scientific, catalog number: BP358-1 )
Tris-Base (Fisher Scientific, catalog number: BP152 )
Ethylenediaminetetraacetic acid, disodium salt, Dihydrate (Fisher Scientific, catalog number: S311 )
Sodium dodecyl sulfate (Fisher Scientific, catalog number: BP166 )
Carboxymethyl cellulose, CMC (Sigma-Aldrich, catalog number: C5678 )
Sterile deionized water (dH2O)
Sterile double distilled water (ddH2O)
Phenol (Fisher Scientific, catalog number: BP226500 )
Chloroform (Fisher Scientific, catalog number: C607-4 )
Isopropanol (Fisher Scientific, catalog number: A451SK-4 )
Ethanol (Fisher Scientific, catalog number: A995-4 )
Primers (Listed below in Table 1)
dNTPs (Sigma-Aldrich, catalog number: DNTP100A-1KT )
Polymerase for PCR (Fisher Scientific, catalog number: FB-6000-10 )
iTaq Univeral SYBR Green Supermix (Bio-Rad, catalog number: 1725122 )
Agarose (Fisher Scientific, catalog number: BP1356 )
Soil mix (Fafard, catalog number: Fafard Growing Mix 2/C-2 )
Peters 20:20:20 General Purpose fertilizer (Hummert International; catalog number: 07-5400-1 )
F. graminearum macroconidia suspension (see Procedure)
F. graminearum mycelial fragments (see Procedure)
Potato Dextrose Agar-Half strength (½ PDA) (see Recipes)
Carboxymethyl Cellulose (CMC) media (see Recipes)
Arabidopsis DNA extraction buffer (see Recipes)
Tris-equilibrated phenol-chloroform (see Recipes)
Spray spore suspension (see Recipes)
Equipment
Hand-held atomizer
Micropipettes (P20, P100 and P1000)
Standard Lab Incubator for cultivating fungus (Fisher Scientific, Fisher ScientificTM IsotempTM)
Plant growth chamber for cultivating Arabidopsis (Percival scientific, model: AR-66L2 )
Thermal cycler (Techne, model: 3PrimeX )
Real-time PCR system (Illumina, EcoTM, catalog number: EC-101-1001 )
Note: This product has been discontinued by the manufacturer.
Compound microscope (Leica, model: DM2000 )
Tabletop centrifuge (Beckman)
Microfuge (Fisher Scientific, Fisher ScientificTM accuSpinTM, model: Micro 17/Micro 17R )
Vortex-Genie 2 (Scientific Industries, catalog number: SI-0236 )
Basic power supply gel electrophoresis powerpack, trays and combs, (Bio-Rad, PowerPacTM, catalog number: 1645050 )
Gel electrophoresis system (Bio-Rad, catalog number: 1704405 )
Software
Variance (ANOVA) (P < 0.05) (SAS Institute Inc, SAS v5.1)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Nalam, V., Sarowar, S. and Shah, J. (2016). Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues. Bio-protocol 6(14): e1877. DOI: 10.21769/BioProtoc.1877.
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Category
Plant Science > Plant immunity > Disease bioassay
Plant Science > Plant immunity > Host-microbe interactions
Microbiology > Microbe-host interactions > Fungus
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1,878 | https://bio-protocol.org/exchange/protocoldetail?id=1878&type=0 | # Bio-Protocol Content
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Peer-reviewed
Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
AL April M. Lewis
SM Silvia S. Matzdorf
KR Kelly C. Rice
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1878 Views: 8346
Edited by: Valentine V Trotter
Reviewed by: Lionel Schiavolin
Original Research Article:
The authors used this protocol in Sep 2014
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The authors used this protocol in:
Sep 2014
Abstract
Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM diacetate). This compound diffuses into cells and intracellular cleavage by esterase enzymes liberates weakly-fluorescent DAF-FM, which reacts with NO or other specific RNS to become highly fluorescent (Kojima et al., 1999). Although quantification of fluorescence is performed using a fluorescent plate reader, it is envisioned that this protocol could be adapted for intracellular NO/RNS imaging of S. aureus biofilms by confocal microscopy. Likewise, this technique could be optimized for the detection of intracellular NO/RNS in other growth conditions (i.e., planktonic cultures) and/or in other bacteria/archaea.
Materials and Reagents
Wrapping film (Fisher Scientific, Parafilm MTM, catalog number: S37440 )
Micro-centrifuge tubes (1.7 ml) (Fisher Scientific, catalog number: 14-222-171 )
Sterile plastic culture tubes (Fisher Scientific, catalog number: 14-956-6A )
Costar 3524 plates (24-well tissue culture treated) (Fisher Scientific, CorningTM CostarTM ,catalog number: 07-200-84 )
Costar 3904 plates (96-well black tissue-culture treated) (Fisher Scientific, CorningTM, catalog number: 07-200-588 )
150-ml Nalgene sterile disposable 0.2 µm filter unit (Fisher Scientific, Thermo ScientificTM NalgeneTM, catalog number: 09-741-01 )
500-ml Nalgene sterile disposable 0.2 µm filter unit (Fisher Scientific, Thermo ScientificTM NalgeneTM, catalog number: 09-741-02 )
S. aureus stock culture, stored at -80 °C in 25% (v/v) sterile glycerol
Glycerol (Fisher Scientific, catalog number: G33-1 )
Tryptic Soy Agar (TSA) plates (BD, BBLTM, catalog number: 221283 )
Tryptic Soy Broth (TSB) (BD, DifcoTM, catalog number: 211825 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-1 )
Dextrose (Fisher Scientific, catalog number: D16-500 )
4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM diacetate) (Thermo Fisher Scientific, catalog number: D-23842 )
Dimethyl sulfoxide (DMSO) (100 ml) (Sigma-Aldrich, catalog number: 276855 )
Human Plasma, lyophilized (5 ml) (Sigma-Aldrich, catalog number: P9523 )
Carbonate-Bicarbonate buffer capsules (Sigma-Aldrich, catalog number: C3041 )
1x Hank's buffered salt solution (HBSS) buffer, containing calcium and magnesium (Fisher Scientific, CorningTM cellgroTM, catalog number: MT21023CV )
Diethylamine (DEA) (100 ml) (Sigma-Aldrich, catalog number: 471216 )
Diethylamine NONOate (DEA/NO) (10 mg vial) (Cayman Chemicals, catalog number: 82100 )
2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide, monopotassium salt (cPTIO) (5 mg vial) (Cayman Chemicals, catalog number: 81540 )
Sterile nuclease-free H2O (Fisher Scientific, InvitrogenTM AmbionTM, catalog number: AM9930 )
NaOH (Thermo Fisher Scientific, catalog number: S318-500 )
50% (v/v) Glycerol Stock Solution(see Recipes)
Biofilm Media (see Recipes)
Carbonate-Bicarbonate Buffer (see Recipes)
20% Human Plasma (see Recipes)
DAF-FM diacetate stock solution (see Recipes)
0.01 M NaOH solution (see Recipes)
DEA/NO stock solution (see Recipes)
cPTIO stock solution (see Recipes)
DAF-FM diacetate working solution (see Recipes)
DEA/NO working solution (see Recipes)
DEA working solution (see Recipes)
Equipment
Multi detection microplate reader (Biotek synergy HT, model: SIAFR )
10S Bio UV/Vis Spectrophotometer (Thermo Fisher Scientific, GENESYSTM, model: 840-208100 )
Plate incubator (VWR International, catalog number: 97058-224 , model: 1556)
Shaking incubator (VWR International, SignatureTM, catalog number: 14004-300 , model: 1570)
Pipet-Lite XLS+ single-channel pipettes (2-20 µl, 20-200 µl, 100-1,000 µl) (Rainin, catalog number: 17014406 )
Rack LTS tips (“P20” 2-20 µl, “P200” 20-200 µl, “P1000” 100-1,000 µl) (Rainin, Green-PakTM and SpaceSaverTM, catalog number: 17001865 , 17001863 and 17001864 )
Labconco Class II A2 Biosafety Cabinet (Fisher Scientific, LabconcoTMPurifierTMLogic+TM, catalog number: 30-238-1100 )
Vortex Mixer (Fisher Scientific, Fisher ScientificTM, catalog number: 02-215-365 )
Micro-centrifuge (Fisher Scientific, Fisher ScientificTM accuSpinTM, catalog number: 13-100-675 )
Chemical Fume Hood
Refrigerator (4 °C)
Freezer (-20 °C)
Freezer (-80 °C)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lewis, A. M., Matzdorf, S. S. and Rice, K. C. (2016). Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus. Bio-protocol 6(14): e1878. DOI: 10.21769/BioProtoc.1878.
Download Citation in RIS Format
Category
Microbiology > Microbial metabolism > Other compound
Microbiology > Microbial signaling > Secondary messenger
Biochemistry > Other compound
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1,879 | https://bio-protocol.org/exchange/protocoldetail?id=1879&type=0 | # Bio-Protocol Content
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Peer-reviewed
Separation of Free and Bound cAMP in Mycobacteria
Arka Banerjee
Sandhya S. Visweswariah
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1879 Views: 7681
Edited by: Valentine V Trotter
Reviewed by: Darrell CockburnJoana Antunes
Original Research Article:
The authors used this protocol in May 2015
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The authors used this protocol in:
May 2015
Abstract
Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of intracellular cAMP may be bound to protein. The methods typically employed to measure cAMP are radioimmunoassay (RIA) and ELISA. However, these procedures include prior acidification of samples that would dissociate cAMP ‘bound’ to protein, and therefore represent the ‘total’ cAMP present in the sample. In this protocol, we describe a method to separate the fraction of cAMP ‘bound’ to protein from what is ‘free’ or not associated with protein. This is performed by subjecting the cytosolic fraction or the culture supernatant to filtration through a membrane with a 3 kDa cut-off. Only ‘free’ cAMP is able to pass through the membrane. Therefore, cAMP concentrations in the filtrate represent the ‘free’ cAMP in the sample. Cyclic AMP levels in the original cytosolic fraction or the culture supernatant represent the ‘total’ cAMP concentration. Subtracting the ‘free’ from the ‘total’ provides the amount of cAMP bound to protein.
Keywords: CAMP CAMP-binding protein Mycobacterium Universal stress protein
Materials and Reagents
15 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339650 )
1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-175-C )
T25 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 707003 )
T75 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 708003 )
Amicon Ultra-0.5 ml 3-kDa centrifugal filter with Ultracel-3 membrane (Merck Millipore Corporation, catalog number: UFC500324 )
50 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339652 )
Glass test tubes for growing 5 and 10 ml cultures.
96 well polystyrene plates (Sigma-Aldrich, product number: M2936 )
0.5 mm diameter glass beads (Bio Spec Products Inc, catalog number: 11079105 )
2 ml polypropylene bead beating vials (Bio Spec Products Inc, catalog number: 10831 )
Mycobacterium smegmatis mc2 155 (ATCC, catalog number: 700084 )
Mycobacterium bovis BCG (ATCC, catalog number: 35734 )
Middlebrook 7H9 medium (BD Biosciences, catalog number: 271310 )
Glycerol (Thermo Fisher Scientific, catalog number: 17904 )
Magnesium sulfate (Merck Millipore Corporation, catalog number: 105886 )
Tween 80 (Sigma-Aldrich, product number: P 4780 )
7H9-Glycerol-Tween 80 medium
Oleic acid/albumin/dextrose/catalase (OADC) growth supplement (BD Biosciences, catalog number: 211886 )
Bradford reagent (Sigma-Aldrich, catalog number: B6916 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144SI-212 )
cAMP ELISA kit (Enzo Lifesciences, catalog number: ADI-900-067 )
cAMP Direct Immunoassay Kit (Abcam plc, catalog number: ab65355 )
Cyclic AMP Competitive ELISA Kit (Thermo Fisher Scientific, catalog number: EMSCAMPL )
Sodium chloride (Merck Millipore Corporation, catalog number: 1064040500 )
Potassium chloride (Merck Millipore Corporation, catalog number: 1049360500 )
Di-Sodium hydrogen phosphate (Merck Millipore Corporation, catalog number: 1065860500 )
Potassium dihydrogen phosphate (Merck Millipore Corporation, catalog number: 1048730250 )
Tris(hydroxymethyl)aminomethane (Merck Millipore Corporation, catalog number: 1083820500 )
2-Mercaptoethanol (Sigma-Aldrich, product number: M6250 )
Phenylmethylsulfonyl fluoride (Sigma-Aldrich, product number: PMSF-RO ROCHE)
Phosphate buffered saline (see Recipes)
Lysis buffer (see Recipes)
Equipment
Note: Biosafety level 2 (BSL 2) is required for culturing M. bovis BCG
37 °C incubator (humidified)/shaker (N-BIOTEK, model: NB-203QR /Eppendorf, New BrunswickTM Innova®, model: 40 )
Spectrophotometer (Eppendorf, BioPhotometer®, model: D30 )
Refrigerated centrifuge (Thermo Fisher Scientific, HeraeusTM MegafugeTM, model: 16R ) equipped with TX-400 Swinging Bucket Rotor (Thermo Fisher Scientific, model: TX-400 ) and adapter (number 20 & 23)
Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17R )
Vortexer (Bio-rad, model: BR-2000 )
Plate reader (reads in the visible spectrum, including 405 and 595 nm) (Tecan, Infinite®, model: m200 PRO )
Bead beater (BioSpec Products, model: Mini-Beadbeater-16 )
Heating block (heats to 95 °C) (NeoLab)
Ultra low temperature freezer (Panasonic, model: MDF-U33V-PE )
Software
GraphPad Prism 5
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Banerjee, A. and Visweswariah, S. S. (2016). Separation of Free and Bound cAMP in Mycobacteria. Bio-protocol 6(14): e1879. DOI: 10.21769/BioProtoc.1879.
Banerjee, A., Adolph, R. S., Gopalakrishnapai, J., Kleinboelting, S., Emmerich, C., Steegborn, C. and Visweswariah, S. S. (2015). A universal stress protein (USP) in mycobacteria binds cAMP. J Biol Chem 290(20): 12731-12743.
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Category
Microbiology > Microbial physiology
Microbiology > Microbial signaling > Secondary messenger
Biochemistry > Other compound > cAMP
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188 | https://bio-protocol.org/exchange/protocoldetail?id=188&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation of Circulating Immune Complexes from TB Patient Serum for Serodiagnosis
Uma Devi Ranganathan
Ramalingam Bethunaickan
AR Alamelu Raja
Published: Vol 2, Iss 11, Jun 5, 2012
DOI: 10.21769/BioProtoc.188 Views: 12916
Original Research Article:
The authors used this protocol in Nov 2006
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The authors used this protocol in:
Nov 2006
Abstract
Estimation of circulating immune complex in tuberculosis patients has shown better insight to the infection. Isolating circulating immune complex helps quantifying both antigen and antibody in the serum. It’s a simple procedure to improve the sensitivity and specificity in serodiagnosis of tuberculosis. This protocol may be modified to detect antigen/antibody in other infectious diseases.
Keywords: Circulating Immune complex Serodiagnosis Tuberculosis TB diagnosis Antigen and antibody detection
Materials and Reagents
Boric acid (Sigma-Aldrich, catalog number: B6768 )
Disodium tetraborate (Borax) (Sigma-Aldrich, catalog number: 71996 )
NaCI (Sigma-Aldrich, catalog number: S3014 )
Polyethylene glycol (PEG 8000) (Sigma-Aldrich, catalog number: 89510 )
Phosphate buffer saline (Life Technologies, Gibco®, catalog number: 20012-027 )
NaHCO3 (Sigma-Aldrich, catalog number: S6014 )
Na2CO3 (Sigma-Aldrich, catalog number: 330361 )
BSA (Sigma-Aldrich, catalog number: A7030 )
Tween 20 (Sigma-Aldrich, catalog number: P9416 )
Antihuman affinipure IgG/lgA/lgM HRP [The Jackson Laboratory, USA, catalog number: 709-036-149 (IgG); 109-036-011 (IgA); 309-036-043 (IgM)]
Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
Citric acid (Sigma-Aldrich, catalog number: 251275 )
O-Phenylenediamine (OPD) (Sigma-Aldrich, catalog number: P5412 )
H2SO4 (Sigma-Aldrich, catalog number: 320501 )
0.1 M borate buffer (pH 8.4) (see Recipes)
7% polyethylene glycol (PEG) (see Recipes)
Phosphate buffer saline (PBS) (see Recipes)
0.06 M Carbonate buffer (pH 9.6) (see Recipes)
Blocking solution (see Recipes)
Primary antibody (see Recipes)
Secondary antibody (see Recipes)
Substrate buffer (see Recipes)
Substrate (see Recipes)
Stop solution (see Recipes)
Equipment
Refrigerated bench-top centrifuges
96 wells flat bottom ELISA plates (Nunc®, catalog number: 44-2404-21 )
ELISA washer (Organon Teknika)
Spectromax ELISA reader (Molecular Devices)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Antibody analysis > Antibody detection
Immunology > Immune cell function > General
Immunology > Host defense > Human
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1,880 | https://bio-protocol.org/exchange/protocoldetail?id=1880&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Ubiquitination Assay for Mammalian Cells
YP Yang Peng
EW Edward Wang
GP Guang Peng
Shiaw-Yih Lin
Published: Vol 6, Iss 14, Jul 20, 2016
DOI: 10.21769/BioProtoc.1880 Views: 23537
Edited by: HongLok Lung
Reviewed by: Yu ChenAndrea Puhar
Original Research Article:
The authors used this protocol in Jan 2015
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The authors used this protocol in:
Jan 2015
Abstract
Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
Keywords: Ubiquitination Mammalian Cells in vivo
Materials and Reagents
10 cm dish
Microcentrifuge tubes
Eppendorf tubes
Agarose beads
U2OS cells
HA-Ubiquitin plasmid
FuGene 6 transfection reagent (Promega Corporation, FuGENE®, catalog number: E2691 )
Reduced-serum medium (Thermo Fisher Scientific, Gibco®, catalog number: 31985-070 )
MG132
1x PBS
Trypsin (Thermo Fisher Scientific, Gibco®, catalog number: 25300 )
IgG control antibody according to the targeted antibody species [e.g., Rabbit IgG (Santa Cruz Biotechnology, catalog number: sc-2027 )]
Agarose conjugate [e.g., Protein A/G agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )]
BRCA1 antibody
RIPA buffer (e.g., Santa Cruz Biotechnology, catalog number: sc-24948 )
Anti-ubiquitin or anti-HA antibody
Tris-HCl
SDS
Bromophenol blue
β-mercaptoethanol
Glycerol
Loading buffer (see Recipes)
Equipment
Tube rotator located in cold room 4 °C
Centrifuges (Eppendorf, model: 5424/5424 R )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
Category
Biochemistry > Protein > Modification
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1,881 | https://bio-protocol.org/exchange/protocoldetail?id=1881&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta
JG Jessica M. Guseman
JN Jennifer L. Nemhauser
BM Britney L. Moss
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1881 Views: 9826
Edited by: Arsalan Daudi
Reviewed by: Eunsook ParkStefanie Rosa
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
An emerging theme in biology is the importance of cellular signaling dynamics. In addition to monitoring changes in absolute abundance of signaling molecules, many signal transduction pathways are sensitive to changes in temporal properties of signaling components (Purvis and Lahav, 2013). The phytohormone auxin regulates myriad processes in plant development. Many of these require the nuclear auxin signaling pathway, in which degradation of the Aux/IAA repressor proteins allows for transcription of auxin-responsive genes (Korasick et al., 2015). Using a heterologous yeast system, we found that Aux/IAAs exhibit a range of auxin-induced degradation rates when co-expressed in isolation with F-box proteins (Havens et al., 2012). Subsequent studies connecting signaling dynamics to plant growth and development confirmed that Aux/IAAs show similar differences in plants (Guseman et al., 2015; Moss et al., 2015). Here, we describe in detail the use of a heat-shock-inducible fluorescence degradation system to capture Aux/IAA degradation in real time in live plant roots. By employing this method, we were able to obtain high Aux/IAA expression and avoid the dampening long term effects of turnover, feedback and silencing. Degradation was dependent on the presence of an Aux/IAA degron and rates increased in response to exogenous auxin.
Keywords: Auxin Fluorescence Degradation Root Aux/IAA
Materials and Reagents
1,000 μl pipet tip (Thermo Fisher Scientific, FisherbrandTM, catalog number: 02-681-4 )
50 ml conical centrifuge tube (Thermo Fisher Scientific, FisherbrandTM, catalog number: 06-443-20 )
1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 )
Square (100 x 100 x 15 mm) Petri plates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 08-757-11A )
Round (35 mm) Petri plates (VWR International, catalog number: 10799-192 )
Micropore tape 1530-0 (3M, catalog number: 70200412230 )
Aluminum foil
Razor blades
Rectangular (24 x 50 mm) microscopy coverslips (Thermo Fisher Scientific, catalog number: 12-544E )
Labeling tape
> 4 ml glass perfume spray bottles (available in craft stores)
Arabidopsis thaliana Columbia-0 (Col) seeds transformed with heat-shock constructs (Guseman et al., 2015; Moss et al., 2015)
Bacto Agar (BD, catalog number: 214010 )
Linsmaier-Skoog Media (Caisson Laboratories, catalog number: LSP03-1LT ) (see Note 1)
Triton X-100 (GE Healthcare, catalog number: US22686 )
Indole-3-acetic acid (IAA) (bioWORLD, catalog number: 705490 )
Kanamycin (Thermo Fisher Scientific, catalog number: BP906-5 )
95% ethanol
Seed sterilization solution (see Recipes)
0.5x Linsmaier-Skoog (LS) liquid media (see Recipes)
Sterile plating agar solution (see Recipes)
0.5x LS media + 0.8% agar (see Recipes)
Cover slip media (0.5x LS Media + 1.2% agar) (see Recipes)
IAA stock solution (5 mM) (see Recipes)
Equipment
Slide warmer (Thermo Fisher Scientific, model: 11-474-470 )
Microscope (Leica Biosystems, model: DMI 3000B ) fitted with a Lumencor SOLA light source and YFP filter cube
Leica long working 40x HCX PL FLUOTAR objective (see Note 2)
Leica camera (Leica Microsystems, model: DFC345 FX )
Forceps (Electron Microscopy Sciences, model: Dumont Tweezers Style 2A )
Timer
Software
Leica LAS AF version 2.6.0 for image acquisition
Fiji/ImageJ software for image analysis
GraphPad Prism6 software package for graphing and statistical analysis
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Guseman, J. M., Nemhauser, J. L. and Moss, B. L. (2016). A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta. Bio-protocol 6(15): e1881. DOI: 10.21769/BioProtoc.1881.
Moss, B. L., Mao, H., Guseman, J. M., Hinds, T. R., Hellmuth, A., Kovenock, M., Noorassa, A., Lanctot, A., Villalobos, L. I., Zheng, N. and Nemhauser, J. L. (2015). Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. Plant Physiol 169(1): 803-813.
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Category
Cell Biology > Cell imaging > Live-cell imaging
Plant Science > Plant biochemistry > Plant hormone
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1,882 | https://bio-protocol.org/exchange/protocoldetail?id=1882&type=0 | # Bio-Protocol Content
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Quantification of Chitinase Activity in Fusarium oxysporum
Carmen Ruiz-Roldan
M. Isabel G. Roncero
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1882 Views: 12725
Edited by: Zhaohui Liu
Reviewed by: Claudia CatalanottiAndreea Daraba
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
Fungal morphogenetic development requires modification and plasticity of the cell wall, which implies synthesis and remodelling of its components, including chitin and glucan. Thus chitinase and glucanase activities are crucial for cell-wall biogenesis and cell division. Quantification of chitinase activity might be useful to identify structural defects that could negatively influence growth and morphogenesis of some filamentous fungi like Fusarium oxysporum, which produces both intracellular and secreted chitinases. The chitinolytic enzymes are categorized based on their enzymatic action on chitin substrates. Endochitinases are defined as the enzymes catalyzing the random cleavage at internal points in the chitin chain. Exochitinases catalyze the progressive release of acetylchitobiose or N-acetylglucosamine from the non-reducing end of chitin, and thus, are referred to as chitobiosidase and β-N-acetylglucosaminidase, respectively. Here we describe a simple method to easily purify chitinases in order to compare both endo- and exo-chitinase activity of different F. oxysporum strains. The protocol can be adapted to any fungal species.
Keywords: Chitinase activity Fusarium Extracellular enzyme Intracellular enzyme
Materials and Reagents
Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15 )
Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
Eppendorf tubes
Sterile spatula (Instrumentación Científica, S.L., catalog number: 2004NE8044 )
Microtiter plates (Thermo Fisher Scientific, catalog number: 2205 )
Dialysis membranes, cut-off 12 kDa (Sigma-Aldrich, catalog number: D9527-100FT )
Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type (J. Tello, Universidad de Almeria, Spain), the Δcon7-1 and the cΔcon7-1 mutant strains (Ruiz-Roldan et al., 2015)
Glycerol (Merck, catalog number: 104092 )
Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483 )
Chitin (Sigma-Aldrich, catalog number: C6137 )
Bio-Rad protein assay Dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 10 735 078 001 )
Polyethylenglycol 35,000 (Sigma-Aldrich, catalog number: 81310 )
Na2HPO4 (Merck, catalog number: 1.06586.0500 )
NaH2PO4 monohydrate (Merck, catalog number: 567549 )
MgSO4·7H2O (Merck, catalog number: 1058865.5000 )
KH2PO4 (Merck, catalog number: 104873.1000 )
KCl (Merck, catalog number: 104933.0500 )
NH4NO3 (Merck, catalog number: 1.01187.1000 )
FeSO4 (Merck, catalog number: 103965.0500 )
ZnSO4·7H2O (Merck, catalog number: 108883 )
MnSO4 monohydrate (Merck, catalog number: 105941 )
Glucose (Coger SAS, catalog number: 24379.363 )
NaCl (Merck, catalog number: 1.06404.5000 )
Fluorimetric Chitinase Assay Kit (Sigma-Aldrich, catalog number: CS1030 ). Contents:
Assay Buffer (25 ml) (Sigma-Aldrich, catalog number: A8730 )
4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (5 mg) (Sigma-Aldrich, catalog number: M2133 )
4-Methylumbelliferyl β-D-N,N’-diacetylchitobioside hydrate (5 mg) (Sigma-Aldrich, catalog number: M9763 )
4-Methylumbelliferyl β-D-N,N’,N’’-triacetylchitotriose (5 mg) (Sigma-Aldrich, catalog number: M5639 )
Chitinase from Trichoderma viride (1 mg) (Sigma-Aldrich, catalog number: C6242 )
4-Methylumbelliferone Standard Solution, 50 mg/ml (1 ml) (Sigma-Aldrich, catalog number: M3570 )
Sodium carbonate (2 g) (Sigma-Aldrich, catalog number: S2127 )
Dimethyl sulfoxide (DMSO) (1 ml) (Sigma-Aldrich, catalog number: D8418 )
0.05 M phosphate buffer (see Recipes)
Synthetic medium (SM) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
Strate stock solutions (20 mg/ml) (see Recipes)
Strate working solutions (see Recipes)
Chitinase control enzyme (see Recipes)
Stop solution (sodium carbonate solution) (see Recipes)
4-Methylumbelliferone standard solution (see Recipes)
Equipment
Hemocytometer (Thoma) (Marienfeld, catalog number: 06 407 10 )
Mini-BeadBeater -16 homogenizer (BioSpec Products)
Mortar and pestle
Orbital incubator with shaking (Infors, model: Multitron Pro )
Microcentrifuge (Eppendorf MiniSpin plus, Fisher Scientific)
Fluorimeter (SpectraFluorPlus, TECAN, catalog number: F129005 )
Water bath (Selecta, catalog number: 6000138 )
Vortex (IKA, model: MS2 Minishaker )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ruiz-Roldan, C. and Roncero, M. I. G. (2016). Quantification of Chitinase Activity in Fusarium oxysporum. Bio-protocol 6(15): e1882. DOI: 10.21769/BioProtoc.1882.
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Category
Biochemistry > Other compound > Chitinase
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1,883 | https://bio-protocol.org/exchange/protocoldetail?id=1883&type=0 | # Bio-Protocol Content
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Neurosphere Co-culture Assay
Fanny Ehret
Steffen Vogler
GK Gerd Kempermann
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1883 Views: 12839
Reviewed by: Manuel Sarmiento
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
The hippocampal niche is one of two areas in the brain where stem cells reside. In this neurogenic niche, stem cells can be found in close proximity to astrocytes and in contact with microvessels consisting of pericytes and endothelial cells. To study the regulatory interplay of this complex niche network in a simplified in vitro model, we established a co-culture system. We investigate the formation of neurosphere under different co-culture conditions by using primary niche cells. Here, we describe the isolation procedure for primary niche cells culture of astrocytes, endothelial cells and pericytes/smooth muscle cells from mouse brain. These niche cells are co-cultured (by hanging inserts) with freshly isolated stem and precursor cells from the adult hippocampus to study the influence of soluble factors. This method is used to investigate factors and cell types regulating stem cell behavior in a niche-like environment.
Keywords: Neuroscience Neurosphere Dentate gyrus Neurogenic niche
Materials and Reagents
Petri dishes (diameter 5.5 cm to 9 cm)
Falcon tubes 50 ml and 15 ml
Eppendorf tubes 2 ml (sterilized by autoclave)
Pasteur pipettes (1 mm diameter)
Brush (da Vinci, model: Nova synthetics series 1570, #3 )
12-well and 24-well tissue culture plates (growth-enhance treated, gamma-sterilised, free of pyrogens, free of DNA/RNA, DNase/RNase)
Filter discs (Sartorius AG, catalog number: FT-3-205-055 )
Falcon cell strainers 40 µm and 100 µm (Thermo Fisher Scientific, catalog number: 08-771-1 and 08-771-19 )
Hanging insert for 24-well plates, 1 µm PET (Merck Millipore Corporation, catalog number: PIRP12R48 )
27G ¾ needle (Braun, StericanTM, catalog number: 4657705 )
Mice at the age of six weeks
Note: 6-8 mice for endothelial and pericyte isolation, at least 1 mouse for neurosphere co-culture assay
Mice at the age of four weeks
Note: 4 mice for astrocyte isolation are needed for one preparation.
Distilled water
Poly-D-lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: P7280 )
Laminin (Sigma-Aldrich, Roche, catalog number: R11243217001 )
70% ethanol
HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175 )
Dextran from Leuconostoc mesenteroides average molecular weight 150,000 (Sigma-Aldrich, catalog number: D4876 )
DMEM/F-12 without glutamine (store at 4 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 21331020 )
DMEM/F-12 with L-glutamine and 15 mM HEPES (store at 4 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 11330032 )
B27 (Thermo Fisher Scientific, GibcoTM, catalog number: 0080085SA )
MCDB131 Media (Thermo Fisher Scientific, catalog number: 10372-019 )
Glutamax (100x stock) (store at 4 °C) (Thermo Fisher Scientific, CTSTM, catalog number: A12860-01 )
Insulin-transferrin-selenium (ITS) premix (store at -20 °C) (Thermo Fischer Scientific, CorningTM, catalog number: CB-40351 )
Non-essential amino acids (100x stock) (store at 4 °C) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11140-035 )
Pen/Strep 100,000 U/ml (-20 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
Heparin (store at 4 °C) (MP Biomedicals, catalog number: 0210193125 )
Collagenase A (Sigma-Aldrich, Roche, catalog number: 10103578001 )
Fetal bovine serum (FBS) (-20 °C) (Merck Millipore Corporation, Biochrom, catalog number: S0613 )
Plasma derived platelet poor serum (Sigma-Aldrich, catalog number: P2918 )
Human EGF (store aliquots at -20 °C) (PEPROTECH, catalog number: AF100-15 )
Human FGF-basic (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-18B )
Human VEGF (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-20 )
Accutase (store at 4 °C) (GENTAUR, PAA Laboratories GmbH, catalog number: L11-007 )
Neuronal tissue dissociation kit (P) (Miltenyl Biotec, catalog number. 130-092-628 )
Trypsin/EDTA (store at -20 °C) (Merck Millipore Corporation, Biochrom, catalog number: L2153 )
PBS with 2% FBS (see Recipes)
30% dextran solution (see Recipes)
Endogrow media (see Recipes)
Astrocyte media (see Recipes)
Pericyte/smooth muscle cell media (see Recipes)
Neurosphere media (see Recipes)
Equipment
Dissection tools: scissors, small spatula, fine curved forceps and scalpel (see Figure 2B)
7 ml Dounce tissue grinder (Capitol Scientific, Wheaton, catalog number: 357542 )
Autoclave
Vacuum pump
Dounce homogenizer
Dissection microscope (Olympus, model: SZ61 )
Inverted microscope (Olympus, model: CKX41 )
Scale plate insert into the eyepiece of the microscope (Olympus)
Cooling centrifuge fitting 2 ml tubes (Eppendorf, model: 5430R )
Swing bucket centrifuge for 15 ml tubes (Eppendorf, model: 5810R )
Incubator at 37 °C with 5% CO2
Tube rotator or orbital shaker
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Ehret, F., Vogler, S. and Kempermann, G. (2016). Neurosphere Co-culture Assay. Bio-protocol 6(15): e1883. DOI: 10.21769/BioProtoc.1883.
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Category
Stem Cell > Adult stem cell > Neural stem cell
Cell Biology > Cell isolation and culture > Cell differentiation
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1,884 | https://bio-protocol.org/exchange/protocoldetail?id=1884&type=0 | # Bio-Protocol Content
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Peer-reviewed
Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells
HS Henrique Borges da Silva
CT Carlos Eduardo Tadokoro
ML Maria Regina D’Império Lima
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1884 Views: 10477
Reviewed by: Pinchas Tsukerman
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Efficient staining methods to identify Plasmodium-infected red blood cells (iRBCs) are crucial to discriminate precisely the immune cells responsible for their elimination from circulation. Here, we describe the protocol for the purification of iRBCs and their subsequent staining with the vital dyes Cell Tracer Violet (CTV) or CellTracker Red CMTPX (CMTPX), both of which readily diffuse into cells and bind covalently to intracellular amines. The iRBCs stained by using this protocol were used in ex vivo phagocytosis assays, to determine the ability of splenic dendritic cells of phagocytizing these parasites (Borges da Silva et al., 2015).
Materials and Reagents
15 ml tubes (TPP, catalog number: 91015 )
1 ml syringes (BD Biosciences, catalog number: 309659 )
B6.Rag2-/- (RAGKO) mice (Jackson Laboratories, C57BL/6)
Plasmodium chabaudi (AS strain)
RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
Fetal bovine serum heat inactivated (Thermo Fisher Scientific, catalog number: 10437028 )
Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070 )
2-mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023 )
Cell Tracer Violet (CTV) (Thermo Fisher Scientific, catalog number: C34557 )
CellTracker Red CMTPX (CMTPX) (Thermo Fisher Scientific, catalog number: C34552 )
Halothane (Sigma-Aldrich, catalog number: H0150000 )
NaCl
KCl
Na2HPO4
KH2PO4
Percoll (GE Healthcare Dharmacon, catalog number: 17089101 )
1x phosphate buffered saline (PBS) (see Recipes)
74% Percoll (see Recipes)
Equipment
Centrifuge (Eppendorf, model: 5804 )
1-ml micropipettes
25 gauge needle (BD Biosciences, catalog number: 305122 )
Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
Epi-Fluorescent microscope (Leica Microsystems, model: DM6000B )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Silva, H. B. D., Tadokoro, C. E. and D’Império-Lima, M. R. (2016). Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells. Bio-protocol 6(15): e1884. DOI: 10.21769/BioProtoc.1884.
Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
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Category
Cell Biology > Cell isolation and culture > Cell isolation
Immunology > Immune cell function > Dendritic cell
Immunology > Immune cell staining > Immunodetection
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1,885 | https://bio-protocol.org/exchange/protocoldetail?id=1885&type=0 | # Bio-Protocol Content
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In vivo DCs Depletion with Diphtheria Toxin and MARCO+/MOMA1+ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice
HS Henrique Borges da Silva
CT Carlos Eduardo Tadokoro
ML Maria Regina D’Império Lima
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1885 Views: 10663
Reviewed by: Pinchas Tsukerman
Original Research Article:
The authors used this protocol in Feb 2015
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Feb 2015
Abstract
To evaluate precisely the relative roles of different splenic phagocytic cells during an immune response, efficient methods for the depletion of specific populations are needed. Here, we describe the protocols for the depletion of splenic dendritic cells (DCs) by human diphtheria toxin (DTx) treatment in target mice (which express the human DTx receptor in all CD11c+ DCs) and for the specific depletion of MARCO+/MOMA-1+ marginal zone macrophages (MZMΦs) with clodronate liposomes (ClLip) treatment (when a small dose of ClLip is ministered, MZMΦs preferentially uptake ClLip, and clodronate is released inside those cells causing apoptosis-mediated cell death). These protocols are adaptations from previous works (Jung et al., 2002; McGaha et al., 2011), and were used to evaluate the respective roles of DCs and of MZMΦs during the acute phase of experimental blood-stage malaria infection (Borges da Silva et al., 2015).
Materials and Reagents
25 gauge needle (BD Biosciences, catalog number: 305122 )
Cell strainer (100 μm pore size) (Corning Incorporated, catalog number: 352360 )
15 ml tubes (TPP, catalog number: 91015 )
1 ml syringes (BD Biosciences, catalog number: 309659 )
1 ml syringes with 30 gauge needle (BD Biosciences, catalog number: 328278 )
B6 mice (Jackson Laboratories, model: B6 )
B6.CD11c-DTR mice (Jackson Laboratories, model: C57BL/6 )
Diphtheria toxin (Sigma-Aldrich, catalog number: D0564 )
Sodium clodronate (Melone Pharmaceutical, catalog number: 22560-50-5 )
RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
Fetal bovine serum heat inactivated (Thermo Fisher Scientific, catalog number: 10437028 )
Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070 )
2-mercaptoethanol Thermo Fisher Scientific, catalog number: 21985023 )
Halothane (Sigma-Aldrich, catalog number: H0150000 )
Monoclonal antibody (mAb) to MARCO (R&D Systems, catalog number: FAB2956P )
mAb to MOMA-1 (Abcam, catalog number: ab51814 )
mAb to F4/80 (eBioscience, catalog number: 47-4801 )
mAb to CD11b (BD Biosciences, catalog number: 562950 )
mAb to CD11c (eBiosciences, catalog number: 17011482 )
mAb to I-Ab (eBiosciences, catalog number: 46532082 )
NaCl
KCl
Na2HPO4
KH2PO4
1x phosphate buffered saline (PBS) (see Recipes)
Staining Buffer (see Recipes)
Supplemented RPMI 1640 (see Recipes)
ClLip (see Recipes)
Equipment
Centrifuge (Eppendorf, model: 5804 )
Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
FACSCanto II Flow Cytometer, 8-color, lasers blue/red/violet (BD Biosciences, catalog number: 338962 )
Push-Pull syringe pump (KD Scientific, Model: KDS120 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Silva, H. B. D., Tadokoro, C. E. and D’Império-Lima, M. R. (2016). In vivo DCs Depletion with Diphtheria Toxin and MARCO+/MOMA1+ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice. Bio-protocol 6(15): e1885. DOI: 10.21769/BioProtoc.1885.
Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
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Category
Immunology > Animal model > Mouse
Immunology > Immune cell function > Dendritic cell
Immunology > Immune cell function > Macrophage
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1,886 | https://bio-protocol.org/exchange/protocoldetail?id=1886&type=0 | # Bio-Protocol Content
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Peer-reviewed
Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases
IQ Isaak Quast
MM Michael A. Maurer
JL Jan D. Lünemann
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1886 Views: 11861
Edited by: Jia Li
Reviewed by: Omar AkilElizabeth V. Clarke
Original Research Article:
The authors used this protocol in Nov 2015
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Nov 2015
Abstract
The immunoglobulin G (IgG) fragment crystallizable (Fc) domain contains a single, highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain, which is buried within the hydrophobic core of each of the two heavy chains. The biantennary core glycan structure, composed of 2 N-acetylglucosamine (GlcNAc) and 3 mannose residues, can be further decorated with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid. Presence or absence of distinct residues can alter IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Here, we provide a protocol for the generation of IgG-Fc de-galactosylated, galactosylated, de-sialylated and sialylated IgG antibodies using recombinant glycosidases and glycosyltransferases.
Keywords: Antibody glycosylation Galactosylation Sialylation Fc glycan ST6Gal
Background
The use of glycosyltransferases for antibody glycan modification allows the attachment of sugar substrates to pre-existing glycan residues. Immunoglobulin G carries a single, highly conserved N-glycosylation site in each of its CH2 domains (Arnold et al., 2007) (Figure 1) allowing site-specific glycan modification with glycosyltransferases. Antibodies may carry additional N-glycans if their Fab domains contain Asn-X-Ser/Thr (X ≠ Pro) sequences (Mellquist et al., 1998). Careful selection of a monoclonal antibody lacking Fab glycosylation is therefore important for Fc-specific glycan modification. The protocol described herein was developed based on the following publications (Kingston, 2003; Kaneko et al., 2006; Anthony et al., 2008; Barb et al., 2009; Quast et al., 2015).
Figure 1. The IgG-Fc N-glycan. Schematic depiction of IgG with two fully processed IgG-Fc N-glycans (left) and composition of the glycan (right).
Materials and Reagents
Amicon Ultra-4 Centrifugal Filter Units with 50 kDa MWCO (Merck Millipore Corporation, catalog number: UFC805096 )
HisTrap HP column (GE Healthcare, catalog number: 29-0510-21 )
StrepTrapTM HP column (GE Healthcare, catalog number: 29-0486-53 )
Empty SPE tubes, 12 ml (Sigma-Aldrich, catalog number: 57176 )
VisidryTM Female Luer Plug (Sigma-Aldrich, catalog number: 57098 )
Caps for 12 ml SPE Tubes (Sigma-Aldrich, catalog number: 52174-U )
15 cm cell culture dish
Spectra/Por 3 dialysis membrane (MWCO 3.5 kD), 45 mm width (VWR International, catalog number: 734-0686 )
10 clamps for dialysis tubing: Spectra/Por Closures, 55 mm (Spectrum, catalog number: 132737 )
0.2 µm syringe filters (Filtropur S plus 0.2) (Sarstedt AG, catalog number: 83.1826.102 )
0.2 µm bottle-top vacuum filter systems (Vakuumfiltration 1000 “rapid” Filtermax) (TTP, catalog number: 99950 )
HEK293, HEK293T or HKB11 cells
MES sodium salt (AppliChem GmbH, catalog number: A3101 )
MES monohydrate (AppliChem GmbH, catalog number: A4730 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
MOPS (AppliChem GmbH, catalog number: A1076 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9451 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 306568 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71380 )
Sodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600-K )
Citric acid (Sigma-Aldrich, catalog number: 251275 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
MnCl2 tetrahydrate (AppliChem GmbH, catalog number: A2087 )
MgCl2 hexahydrate (AppliChem GmbH, catalog number: A4425 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
Sodium dihydrogen phosphate monohydrate (NaH2PO4) (AppliChem GmbH, catalog number: A1047 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Trizma® hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941-500 G )
Glycine (Sigma-Aldrich, catalog number: G8898 )
Imidazole (AppliChem GmbH, catalog number: A1073 )
Tween 20 (Sigma-Aldrich, catalog number: P7949 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Sodium azide (AppliChem GmbH, catalog number: A1430 )
D(+)-Lactose monohydrate (AppliChem GmbH, catalog number: A0880 )
1 M Tris, pH 8.8 (AppliChem GmbH, catalog number: A4265 )
d-Desthiobiotin (Sigma-Aldrich, catalog number: D1411-500 MG )
Penicillin and streptomycin, liquid (Thermo Fischer Scientific, GibcoTM, catalog number: 15070-063 )
UDP-galactose (Merck Millipore Corporation, Calbiochem®, catalog number: 670111-50 MG )
CMP-sialic acid (Merck Millipore Corporation, Calbiochem®, catalog number: 233264-5 MG )
StarGate® pCSG-IBA-144 Acceptor Vectors (IBA, catalog number: 5-5144-001 )
StarGate® transfer reagent kit (IBA, catalog number: 5-1603-001 )
Western Blocking Reagent, Solution (Sigma-Aldrich, catalog number: 11921673001 )
Neuraminidase from Clostridium perfringes, recombinantly expressed in E. coli (New England Biolabs, catalog number: P0720S )
β1-4-galactosidase from Streptococcus pneumoniae, recombinantly expressed in E. coli (Merck Millipore Corporation, Calbiochem®, catalog number: 345806-50 MIU )
DMEM high glucose (4.5 g/L) (Thermo Fischer Scientific, GibcoTM, catalog number: 11965092 )
Protein G sepharose for fast flow (GE healthcare, catalog number: 17-0618-01 )
20 µM polyethylene (PE) frits (Sigma-Aldrich, catalog number: 57181 )
Biotinylated Lens Culinaris agglutinin (LCA) (Reactolab, catalog number: B-1045 )
Biotinylated Erythrina Cristagalli lectin (ECL) (Reactolab, catalog number: B-1145 )
Biotinylated Elderberry bark lectin (Sambucus nigra agglutinin, SNA) (Reactolab, catalog number: B-1305 )
Agarose bound Sambucus Nigra Lectin (SNA), Vector Laboratories (Reactolab, catalog number: AL1303 )
Streptavidin-HRP (Mabtech, catalog number: 3310-9 )
MEM NEAA (non-essential amino acids) 100x (Thermo Fischer Scientific, GibcoTM, catalog number: 1140050 )
Sodium pyruvate solution (100 mM) (Sigma-Aldrich, catalog number: S8636 )
PierceTM ECL Western Blotting Substrate (Thermo Fischer Scientific, catalog number: 32106 )
SuperSignalTM West Pico Chemiluminescent Substrate (Thermo Fischer Scientific catalog number: 34080 )
General
Tris-buffered saline (TBS) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
For purification of recombinant glycosyltransferases
293, 293T and HKB11 cell culture medium (see Recipes)
293, 293T and HKB11 cell protein expression medium (see Recipes)
2x HEPES-buffered saline (HeBS) (see Recipes)
2.5 M CaCl2 (see Recipes)
Strep-trap column elution buffer (see Recipes)
His-trap elution buffer (see Recipes)
His-trap wash buffer (see Recipes)
His-trap supernatant dilution buffer (see Recipes)
For enzymatic galactosylation
Galactosyltransferase buffer (see Recipes)
For enzymatic sialylation
Sialyltransferase buffer (see Recipes)
For enrichment of highly-sialylated antibodies using SNA-agarose
SNA-agarose binding/wash buffer (see Recipes)
SNA-agarose elution Buffer (see Recipes)
SNA-agarose clearing Buffer (see Recipes)
0.08% NaN3 (see Recipes)
For de-sialylation using recombinant neuraminidase
Neuraminidase buffer (see Recipes)
For de-galactosylation using recombinant β1-4-galactosidase
β1-4-galactosidase buffer (see Recipes)
For purification of glyco-modified antibodies using gravity flow protein-G sepharose columns
Protein G column elution buffer (see Recipes)
Protein G eluate neutralization buffer (see Recipes)
For analysis of antibody glycosylation by lectin-blotting
Lectin blot incubation buffer (see Recipes)
Lectin blot wash buffer (see Recipes)
Equipment
Spectrophotometer (Thermo Scientific, NanoDrop, model: 1000 )
Tabletop centrifuge capable of > 20,000 x g centrifugation and cooling to 4 °C (Eppendorf)
GE ÄKTAprime plus (GE Healthcare, catalog number: 11-0013-13 )
Mini-PROTEAN® Tetra vertical electrophoresis cell, 4-gel, for 1.0 mm thick handcast gels, with PowerPacTM Basic power supply (Bio-Rad, catalog number: 1658025FC )
Fusion FX7 gel imaging system (Vilber, model: Fusion FX7 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Quast, I., Maurer, M. A. and Lünemann, J. D. (2016). Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases. Bio-protocol 6(15): e1886. DOI: 10.21769/BioProtoc.1886.
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Category
Molecular Biology > Protein > Protein-protein interaction
Immunology > Antibody analysis > Antibody modification
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1,887 | https://bio-protocol.org/exchange/protocoldetail?id=1887&type=0 | # Bio-Protocol Content
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A Highly Efficient Method for Measuring Oxygen Consumption Rate in Fusarium graminearum
DG Daniel Gebhard
JB Jakob Bönnighausen
JB Jörg Bergemann
WS Wilhelm Schäfer
Jörg Bormann
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1887 Views: 9601
Edited by: Valentine V Trotter
Reviewed by: Emilia Krypotou Jose Thekkiniath
Original Research Article:
The authors used this protocol in Dec 2015
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Abstract
The filamentous ascomycete Fusarium graminearum is the causal agent of Fusarium head blight, a devastating disease of cereals with a worldwide distribution. Fusarium graminearum infections result in a quantitative yield reduction by impairing the growth of the kernels, and a qualitative reduction by poisoning the remaining kernels with mycotoxins toxic to animals and humans. The colonization of wheat florets by phytopathogenic fungus requires high-efficiency energy generation in the mitochondria (Bönnighausen et al., 2015). Mitochondrial activity in microorganisms can be measured using the oxygen consumption rate (OCR) method. Here we describe a method for the assessment of fungal respiration using an XF24 extracellular flux analyzer. The Seahorse XF Analyzer is a microplate-based respirometer which measures oxygen consumption by changes in the fluorescence of immobilized fluorophores (Gerencser et al., 2009). Multiple mitochondrial parameters can be measured by the application of mitochondrial substrates and inhibitors which are injected automatically during the assays via ports (Divakaruni et al., 2014). The experimental work-flow involves the inoculation with conidia and the application of specific inhibitors of mitochondrial functions. The analysis of fungal respiration represents a valuable tool that complements classical phenotypic screenings.
Materials and Reagents
Pipette tips
Conidia of F. graminearum (preferably fresh, not frozen)
Seahorse XF Cell Mito Stress Test Kit including oligomycin, rotenone, antimycin A and Cyanide-p-trifluoromethoxyphenyl hydrazone (Seahorse Bioscience, catalog number: 103015-100 )
Seahorse XF24 Islet FluxPak containing XF24 cartridges and Islet capture microplates (Seahorse Bioscience, catalog number: 101174-100 )
Calibrant solution (Seahorse Bioscience, catalog number: 103059-000 )
Ca(NO3)2·4H2O (Carl Roth, catalog number: X886.1 )
KH2PO4 (Carl Roth, catalog number: P018.1 )
MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391-500G )
NaCl (Carl Roth, catalog number: 9265.1 )
Sucrose
H3BO3 (Carl Roth, catalog number: 6943.1 )
CuSO4·5H2O (Sigma-Aldrich, catalog number: 209198-250G )
KI (Carl Roth, catalog number: 6750.1 )
MnSO4·H2O (Carl Roth, catalog number: 7347.2 )
(NH4)6Mo7O24·4H2O (Sigma-Aldrich, catalog number: 09880-100G )
ZnSO4·7H2O (Carl Roth, catalog number: 7316.1 )
FeCl3·6H2O (Carl Roth, catalog number: 7119.1 )
Solution A (see Recipes)
Solution B (see Recipes)
Suspension D (see Recipes)
Minimal medium (see Recipes)
Equipment
Multi-channel pipettes (Sigma-Aldrich, catalog number: Z683930-1EA )
XF24 extracellular flux analyzer (Seahorse Bioscience, model: Seahorse XFe24 )
Incubator at 28 °C (Heraeus B20/UB20) (Thermo Fisher Scientific, catalog number: 50061005 )
Microplate centrifuge (Eppendorf, catalog number: 022620509 )
Software
XF24 Extracellular Flux Analyzer Software
Spreadsheet software program [e.g., Excel (Microsoft)]
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gebhard, D., Bönnighausen, J., Bergemann, J., Schäfer, W. and Bormann, J. (2016). A Highly Efficient Method for Measuring Oxygen Consumption Rate in Fusarium graminearum. Bio-protocol 6(15): e1887. DOI: 10.21769/BioProtoc.1887.
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Category
Microbiology > Microbial physiology > Respiration
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1,888 | https://bio-protocol.org/exchange/protocoldetail?id=1888&type=0 | # Bio-Protocol Content
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Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
SD Steffen Lorenz Drees
Mathias Lübben
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1888 Views: 9910
Edited by: Valentine V Trotter
Reviewed by: Esteban Paredes-Osses
Original Research Article:
The authors used this protocol in Aug 2015
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Abstract
Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties of these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to predict. We established a method suitable to probe metal transfer between two proteins, provided the proteins are amenable to purification and in vitro handling. It consists of the loading with metals, the co-incubation and the separation of metal-exchanging proteins with subsequent determination of bound metal content. The method is exemplified by experimental data of ours probing the transfer of copper(I) between the membrane-extrinsic metal binding domain MBD2 and the transmembrane domain of CopA, a copper export ATPase from Escherichia coli (Drees et al., 2015).
Keywords: Copper-binding protein Copper chaperone Heavy metal associated domain Copper transporter P-type ATPase
Materials and Reagents
Desalting NAP5 columns (e.g., GE Healthcare, HiTrap Desalt, catalog number: 17-0853-01 )
Gel filtration columns
We used prepacked GE 10/300 Tricorn columns, a Superdex 75 (GE Healthcare, catalog number: 17-5174-01 ) for small soluble proteins, or a Superose 6 for lipid-reconstituted samples (GE Healthcare, catalog number: 17-5172-01 ).
Proteins
Notes:
Metal binding proteins of interest must be on the hand in purified native state, with aggregates having been removed prior to the experiments.
Proteins should be available in milligram quantities and should preferably have different molecular weights (which is a prerequisite for gel filtration).
Proteins too small to be distinguished from each other on gel filtration columns could be fused to a polypeptide tag, e.g., the maltose binding protein or glutathione-S-transferase in order to artificially increase the molecular weight difference of the metal ion transfer pair.
We used an eGFP tag because it is relatively inert in redox-reactions and facilitates easy detection and concentration determination.
Care has to be taken with the use of a His-tag for means of protein purification. Although it was demonstrated that His-tags do not interfere with copper binding and quantification experiments under certain conditions (González-Guerrero et al., 2008), the metal chelating property of the hexahistidine sequence may cause metal carry-overs in some experiments.
3-(N-morpholino) propanesulfonic acid (MOPS), buffer grade (Sigma-Aldrich, catalog number: M1254 )
Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
Sodium ascorbate (Sigma-Aldrich, catalog number: 11140 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
Phosphatidylcholine (PC) (Sigma-Aldrich, catalog number: P3644 )
Sodium dodecylsulfate (SDS) (Sigma-Aldrich, catalog number: L6026 )
Bathocuproinedisulfonate (BCS) (Sigma-Aldrich, catalog number: B1125 )
Bicinchoninic acid (BCA) (Sigma-Aldrich, catalog number: D8284 )
β-dodecylmaltoside (β-DDM) (Sigma-Aldrich, catalog number: D5172 )
Copper sulfate (CuSO4) (Sigma-Aldrich, catalog number: 451657 )
Buffer A (see Recipes)
BCA-copper buffer (see Recipes)
SDS solution (see Recipes)
Ascorbate solution (see Recipes)
BCS solution (see Recipes)
Equipment
(Optional) A dynamic light scattering instrument [e.g., High performance particle sizer (Malvern Instruments, model: HPP 5001 or Zetasizer Nano S )]
Ultrasonic homogenizer for preparation of lipid emulsions (e.g., BANDELIN electronic GmbH & Co, model: UP 400 S )
Ultrasonic bath for buffer degassing (e.g., BANDELIN electronic GmbH & Co, model: Sonorex DL510H )
An automated chromatography unit (e.g., GE Healthcare, Äkta Purifier or Explorer)
Note: It is essential for the experiments. Use of a regular HPLC unit with fraction collector would be just as fine, provided that some modifications with respect to the tolerance of salt-containing elution buffers have been taken care of.
A thermostated shaking incubator for 1.5 ml type reaction vials (e.g., Eppendorf, catalog number: 5384000012 )
Centrifugal evaporator (e.g., Eppendorf, catalog number: 5305000304 )
Calibrated micropipette (e.g., Eppendorf, catalog number: 3120000062 ), alternatively: analytical balance (e.g., Mettler-Toledo International Inc., catalog number: 11142056 )
UV-VIS Spectrophotometer (e.g., Jasco, model: J-650 ) and Nanodrop spectrophotometer (Thermo Fisher Scientific), alternatively: UV-VIS spectrophotometer and small volume cuvette (e.g., Hellma, model: 105.250-QS or Eppendorf, µCuvette®, model: G1.0 )
Quartz microcuvette (e.g., Hellma, catalog number: 104-10-40 104-QS )
Standard SDS-PAGE equipment (e.g., Bio-Rad Laboratories, catalog number: 1658001FC )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Drees, S. L. and Lübben, M. (2016). Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins. Bio-protocol 6(15): e1888. DOI: 10.21769/BioProtoc.1888.
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Category
Biochemistry > Protein > Interaction
Microbiology > Microbial biochemistry > Protein
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1,889 | https://bio-protocol.org/exchange/protocoldetail?id=1889&type=0 | # Bio-Protocol Content
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Acute Live/Dead Assay for the Analysis of Toxic Effects of Drugs on Cultured Neurons
NS Noa Sadeh
EO Efrat Oni-Biton
Menahem Segal
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1889 Views: 12814
Edited by: Oneil G. Bhalala
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
The primary culture of central nervous system (CNS) neurons is a popular test system for a rapid, quantitative and reliable assessment of the effects of drugs on central neurons. Consequently, studies on the excitotoxicity of NMDA activation and on intracellular calcium handling machineries with respect to ischemic damage to the brain as well as neurodegenerative diseases have been highly productive (Ankarcrona et al., 1995). This created the need to establish a standard method for assessment of neurotoxicity. Several methods are currently being used, including LDH leakage and MTT assays (Mosmann, 1983; Decker and Lohmann-Matthes, 1988). We have used another common method for assessing acute cell death, the dead/live assay (Slepian et al., 1996). It provides a precise time and concentration evaluation of the process of cell death following exposure to a toxic substance, in our case, zeta-inhibitory peptide (ZIP), previously proposed to act as a selective PKM-zeta antagonist (Ling et al., 2002; Pastalkova et al., 2006; Sadeh et al., 2015). In this assay, we load cells with Calcein-AM, which, upon penetration into live neurons, is converted from a non-fluorescent compound into a highly fluorescent green fluorophore. Subsequently, we expose the neurons to different concentrations of ZIP for various durations, in the presence of propidium iodide (PI) which penetrates dead cells, and count red/green fluorescent cells. This method allows us to examine which cells were alive before, and died after exposure to the toxic substance as well as the time course of cell death.
Keywords: Cultured hippocampal neuron Zeta inhibitory peptide Live/dead imaging Calcein Propidium iodide
Materials and Reagents
24 well plates (Nunc)
Rat pups age P0, hippocampus (Figure 1)
L-15 (Thermo Fisher Scientific, Gibco®, catalog number: 21083 )
Glucose (Sigma-Aldrich)
Gentamicin (Sigma-Aldrich, catalog number: G1272 )
Trypsine (Thermo Fisher Scientific, Gibco®, catalog number: 150900046 )
DNase
Enriched MEM (Minimal Essential Medium, enriched with glucose (0.6%), Glutamax (2 mM), and gentamicin (15 μg/ml) (Thermo Fisher Scientific, Gibco®, catalog number: 32360-026 )
B-27 (Thermo Fisher Scientific, Gibco®, catalog number: 17504 )
Horse serum (Thermo Fisher Scientific, Gibco®, catalog number: 16050 )
Fetal calf serum (Gibco)
5-Fluoro-2-deoxyuridine (FUDR) (Sigma-Aldrich, catalog number: F0503 )
Calcein-AM (Sigma-Aldrich)
Zeta inhibitory peptide (ZIP) (AnaSpec)
Scr-ZIP (Anaspec, same amino acids as ZIP, in a scrambled order)
Propidium iodide (Sigma-Aldrich)
NaCl (Sigma-Aldrich)
KCl
MgCl2 (Sigma-Aldrich)
CaCl2 (Sigma-Aldrich)
HEPES (Sigma-Aldrich, catalog number: 83264 )
Standard extracellular medium (see Recipes)
Figure 1. 3D reconstruction of a rodent brain. In green-bilateral hippocampus (reproduced from Allen Brain Atlas)
Equipment
Biological hood (Haroshet Ltd.)
37 °C/5% CO2 humidified incubator (Tuttnauer, model: TUTT2424-2 )
Confocal laser scanning inverted microscope (Zeiss, model: LSM 510 ), with a 40x oil-immersion objective (1.3 NA)
Standard desktop centrifuge
Inverted microscope (Nikon) equipped with phase optics
Dissecting binocular microscope
Software
ImageJ
LSM 510 software
Procedure
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Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Cell viability > Cell death
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189 | https://bio-protocol.org/exchange/protocoldetail?id=189&type=0 | # Bio-Protocol Content
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Peer-reviewed
Development of T Cells through Co-culture Lymphoid Progenitor Cells with OP9-DL1 Stromal Cells in vitro
HW Hongcheng Wang
Published: Vol 2, Iss 10, May 20, 2012
DOI: 10.21769/BioProtoc.189 Views: 20850
Original Research Article:
The authors used this protocol in Sep 2009
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Sep 2009
Abstract
Scientists commonly study the development of lymphocytes in two ways, adoptive transfer of hematopoietic stem cells or progenitor cells into recipient mice and fetal thymic organ culture (FTOC). Both strategies, especially the first one, are still widely used. However there are some limitations of these two methods such as being time consuming, resulting in limited cell yield, and challenges in the technology. During the last decade, OP9 stromal cells co-culture system has been modified to support lymphocyte development in vitro. This alternative way offers researchers a simple, efficient approach to support lymphoid progenitors to develop in vitro. A more important advantage of this system is that a lot of factors involved in lymphocyte development, such as cytokines and Notch signaling pathway, can be manipulated in order to delineate the mechanisms more clearly. This protocol is based on the experience of supporting T cell development by OP9-DL1 stromal cells. As a matter of fact, this system can be used to facilitate other lymphocytes development in vitro, such as B cells and NK cells, depending on the type of stromal cells and different combinations of cytokines.
Stromal cell line, OP9, was derived from the bone marrow of op/op mice that are deficient for macrophage-colony stimulating factor (M-CSF). In order to investigate the role of Notch signaling on the differentiation of T cells in vitro, intensive studies have been recently done through the co-culture system including lymphoid progenitor cells and modified OP9 stromal cells that express Notch ligands such as Delta-like 1, Delta-like 4, or control vector. These derived OP9 stromal cells lines are termed as OP9-DL1, OP9-DL4, and OP9-V or OP9-vector, respectively.
Materials and Reagents
Cell line: OP9-DL1 stromal cells (OP9-DL1 stromal cells originally made by Dr. J. C. Zuniga-Pflucker. Individual researchers can request this cell line directly from him or from others who got the cell line from him)
OP9-DL1 Recombinant mouse IL-7 (R&D Systems, catalog number: 407-ML-005 )
Recombinant mouse Flt-3 ligand (R&D Systems, catalog number: 427-FL-005 )
Lymphoid progenitor cells
Alpha medium/ Minimum essential medium (MEM) (Life Technologies, Gibco™, catalog number: 41061-037 )
Fetal bovine serum (FBS)
Penicillin-streptomycin-glutamine (Cellgro, catalog number: 30-009-CI )
2-Mercaptoethanol (Thermo Fisher Scientific, catalog number: ICN19024290 )
Phosphate buffered saline (PBS)
0.5 M EDTA (Life Technologies, Ambion®, catalog number: AM9260G )
Culture medium (see Recipes)
Equipment
Centrifuges
10 cm tissue culture dish
96-well plate, 48-well plate, or 24-well plate
Cell culture Incubator
15 ml centrifuge tube
Incubator
42 or 80 µm filter
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wang, H. (2012). Development of T Cells through Co-culture Lymphoid Progenitor Cells with OP9-DL1 Stromal Cells in vitro. Bio-protocol 2(10): e189. DOI: 10.21769/BioProtoc.189.
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Category
Immunology > Immune cell function > Lymphocyte
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1,890 | https://bio-protocol.org/exchange/protocoldetail?id=1890&type=0 | # Bio-Protocol Content
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Peer-reviewed
Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120
Kangming Chen
Huilan Zhu
Liping Gu
Shengni Tian
Ruanbao Zhou
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1890 Views: 10001
Edited by: Maria Sinetova
Reviewed by: Claudia CatalanottiYoko Eguchi
Original Research Article:
The authors used this protocol in Jun 2015
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Abstract
Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N2-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an integrative vector to produce a cargo plasmid. Upon a single crossover (homologous recombination) between the cargo plasmid and the Anabaena chromosome, the endogenous target gene is disrupted by generating 3’- and 5’-deleted fragments. This gene inactivating protocol is based on an integrative vector pZR606 (Chen et al., 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.
Keywords: Genetic tools for bacteria Genetic engineering Integration vector method Single cross-over method Synthetic cyanobacteria
Materials and Reagents
Petri dishes (Fisher Scientific, catalog number: FB0875713 )
Glass flask (Fisher Scientific, catalog number: FB-500-50 )
1.5 ml centrifuge tubes (Fisher Scientific, catalog number: 02-682-550 )
15 ml conical centrifuge tubes (Fisher Scientific, catalog number: 05-527-90 )
50 ml conical centrifuge tubes (Fisher Scientific, catalog number: 12-565-270 )
Anabaena sp. strain PCC 7120 (hereafter Anabaena 7120)
Escherichia coli (E. coli) NEB10β [Δ(ara-leu) 7697 araD139 fhuAΔlacX74 galK16 galE15 e14-Φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL(StrR)rph spoT1Δ(mrr-hsdRMS-mcrBC); New England BioLabs, catalog number: C3019H ]
E. coli HB101 [F-mcrB mrr hsdS20(rB-mB-) recA13, leuB6, ara-14, proA2, lacY1, galK2, xyl-5, mtl-1, rpsL20(SmR) glnV44 λ- ; Promega, catalog number: L2015 ]
Conjugal plasmid pRL443 and helper plasmid pRL623 (Elhai et al., 1997)
pZR606, an integrative vector for Anabaena 7120 (Chen et al., 2015). pZR606 is available upon request (GenBank, catalog number: KJ500179.1 )
pZR670 (available upon request), a replicative vector in Anabaena 7120 (Xu et al., 205) [The map of pZR670 is provided (Figure S1).]
LB broth (Sigma-Aldrich, catalog number: L3522-1KG )
LB agar, used for growing E. coli (MP, catalog number: 100262 )
Note: This brand of agar does not work for cyanobacteria.
Agar, required for growing Anabaena 7120 and other cyanobacteria (Fisher Scientific, catalog number: A360-500 )
Immobilon-NC transfer membrane (Millipore, catalog number: HATF08550 )
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
Chloramphenicol (Fisher Scientific, catalog number: BP904-100 )
Erythromycin (Sigma-Aldrich, catalog number: E6376-25G )
Kanamycin sulfate (Sigma-Aldrich, catalog number: K4000-25G )
Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S9007-25G )
MgSO4·7H2O (MP, catalog number: 194833 )
CaCl2·2H2O (Fisher Scientific, catalog number: BP510-500 )
NaCl (Fisher Scientific, catalog number: S271-1 )
K2HPO4 (Fisher Scientific, catalog number: BP363-500 )
KNO3 (Fisher Scientific, catalog number: BP368-500 )
NaNO3 (Fisher Scientific, catalog number: BP360-500 )
MnCl2·4H2O (Fisher Scientific, catalog number: M87-100 )
Na2Mo4·2H2O (99% purity) (Acros Organics, catalog number: 206371000 )
ZnSO4·7H2O (Fisher Scientific, catalog number: Z76-500 )
CuSO4·5H2O (Fisher Scientific, catalog number: BP346-500 )
H3BO3 (Fisher Scientific, catalog number: BP168-500 )
NH4VO3 (Acros Organics, catalog number: 194910500 )
CoCl2·6H2O (Fisher Scientific, catalog number: C371-100 )
KOH (Fisher Scientific, catalog number: P250-500 )
Na2EDTA·2H2O (Fisher Scientific, catalog number: BP120-500 )
FeSO4·7H2O (Fisher Scientific, catalog number: I146-500 )
Allen and Arnon medium plus nitrate: AA/8(N) (see Recipes)
Equipment
Centrifuges (Beckman, model: Allegra X-15R ; Thermo Fisher Scientific, SorvallTM LegendTM, model: Micro17 )
Innova-44R shaker equipped with continuous fluorescent light illumination (ca. 50-100 μE/m2 s) (Eppendorf, New BrunswickTM Innova®, model: 44R )
Cyanobacteria Culture Room (constant at 30 °C) equipped with a digital temperature controlling system and Sylvania fluorescent light bulbs (F40CWX, 40W/4100K, T12)
Note: Light shelves in cyanobacteria culture room are able to provide continuous light illumination (ca. 50-150 μE/m2 s).
ESCO laminar flow cabinet (ESCO, Clean BenchAirstream® model: AHC-4B2 )
Water bath sonicator (Bransonic® Ultrasonic Cleaner, model: 1510R-MT )
Microscope, Olympus upright compound microscope (Olympus, model: AX70; BX53 )
PCR thermal cycler (Bio-Rad Laboratories, TouchTM, model: C1000 )
UV-Vis spectrophotometer (Thermo Fisher Scientific, model: GeneSyS 10S )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, K., Zhu, H., Gu, L., Tian, S. and Zhou, R. (2016). Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120. Bio-protocol 6(15): e1890. DOI: 10.21769/BioProtoc.1890.
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Category
Molecular Biology > DNA > Transformation
Microbiology > Microbial genetics > Transformation
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1,891 | https://bio-protocol.org/exchange/protocoldetail?id=1891&type=0 | # Bio-Protocol Content
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Peer-reviewed
EST-SSR Analysis and Cross-species Transferability Study in Lavandula
Ayelign M. Adal
ZD Zerihun A. Demissie
SM Soheil S. Mahmoud
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1891 Views: 9350
Edited by: Samik Bhattacharya
Reviewed by: Yi Zhang
Original Research Article:
The authors used this protocol in Apr 2015
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Abstract
The genus Lavandula comprises of several economically important lavender species that are mainly cultivated worldwide for essential oil production. Identification of lavender species and their cultivars has been a huge bottleneck in lavender industries due to lack of appropriate identification mechanisms. Recent advances in modern technologies would help to address these identification issues through development of potential molecular markers, including simple sequence repeats (SSRs). SSRs can be developed from specific species, and can be potentially used for related species, which lack the source sequences to develop species-specific SSRs. Here, we describe the guidelines and steps of identifying and analyzing SSRs from expressed sequence tag (EST) sequences of lavender species. We also detail the validation procedures of selected EST-SSRs in distinguishing source (donor) species as well as related species.
Keywords: EST-SSR Lavandula SSR Lavender Essential oil
Materials and Reagents
1.5 ml centrifuge tube
1-200 µl volume pipette tips
Leaf tissue (Okanagan Lavender and Herb Farm and The Greenery Garden Center, Kelowna, BC, Canada)
DNA sequences (expressed sequence tag) (EST)
Liquid N2 (Praxair)
Genomic DNA Mini kit (Plant) GP100 (Geneaid Biotech Ltd., catalog number: GP100 )
10x PCR buffer (NEB, catalog number: M0320S )
Taq polymerase (NEB, catalog number: M0320S )
dNTPs (Omega Bio-Tek, catalog number: TQAC135 )
MgCl2 solution (NEB, catalog number: M0320S )
Bovine serum albumin (BSA) (NEB, catalog number: B9000S )
Custom-synthesized primers (Thermo Fisher Scientific, InvitrogenTM)
SYBR® Safe DNA gel staining (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
10x DNA loading dye (Ward’s Science, catalog number: 389115 )
Acrylamide 40% solution (Acrylamide: Bis-Acrylamide 19:1) (Thermo Fisher Scientific, catalog number: BP1406-1 )
50 bp DNA ladder (NEB, catalog number: N3236S )
1 kb DNA ladder (FroggaBio, catalog number: DM010-R500 )
Nuclease-free water (Thermo Fisher Scientific, catalog number: BP5611 )
Ammonium persulfate (APS) (Thermo Fisher Scientific, catalog number: BP179-100 )
NNN'N'-tetramethyl-ethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
Tris Base (Thermo Fisher Scientific, catalog number: BP152-1 )
Glacial acetic acid (Thermo Fisher Scientific, catalog number: 351272-212 )
Na2EDTA (VWR International, catalog number: CA71007-124 )
Boric acid (Thermo Fisher Scientific, catalog number: B168-1 )
PCR reaction mix (see Recipes)
0.5 M EDTA (see Recipes)
50x TAE buffer (see Recipes)
1x TAE buffer (see Recipes)
5x TBE buffer (see Recipes)
0.5x TBE working solution (see Recipes)
10% Ammonium persulfate (APS) (v/w) (see Recipes)
1 M Tris (see Recipes)
10 mM Tris buffer, pH 8.0 (see Recipes)
1% agarose gel (Thermo Fisher Scientific, catalog number: BP160-500 ) (see Recipes)
6% polyacrylamide gel (see Recipes)
Equipment
Desktop computer (Dell, model: precision T3610 Tower Workstation )
Mortar and pastel (VWR International, Porcelain)
Balance (Shimadzu Corporation, model: ELB300 )
Freezer (-20 °C) (Frigidaire, model: FFFH20F2QW )
Thermocycler (Thermo Fisher Scientific, ABI Applied BiosystemTM, model: Veriti® 96-Well Thermal Cycler )
Horizontal gel electrophoresis (Thermo Fisher Scientific, OwlTM EasyCastTM, model: B2 Mini )
Vertical gel electrophoresis (Bio-Rad Laboratories, model: Mini-protein Tetra system )
Microcentrifuge (Eppendorf, model: 5417C )
Microwave oven (Danby Designer)
Water bath (Thermo Fisher Scientific, IsotempTM Digital-Control, model: 210 )
Gel Imager (Mandel Scientific, model: Kodak Gel Logic 440 )
Sterilmatic Autoclave (Thomas Scientific, model: STME-L )
Spectrophotometer (Thermo Scientific, NanoDropTM, model: 2000 )
Software
BatchPrimer 3 (http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi) (You et al., 2008)
SSR mining server (https://www.rosaceae.org/bio/content?title=&url=/cgi-bin/gdr/gdr_ssr) (Jung et al., 2008)
Perl script MIcroSAtelitte (MISA) identification tool (http://pgrc.ipk-gatersleben.de/misa/)
Note: optional software, but not used in our study
Blast2go online platform (https://www.blast2go.com/) (Conesa et al., 2005)
OligoAnalyzer 3.1 (https://www.idtdna.com/calc/analyzer)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Adal, A. M., Demissie, Z. A. and Mahmoud, S. S. (2016). EST-SSR Analysis and Cross-species Transferability Study in Lavandula. Bio-protocol 6(15): e1891. DOI: 10.21769/BioProtoc.1891.
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Category
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > Genotyping
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1,892 | https://bio-protocol.org/exchange/protocoldetail?id=1892&type=0 | # Bio-Protocol Content
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Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
TR Thibaud T. Renault
Mark P.A Luna-Vargas
Jerry E. Chipuk
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1892 Views: 11731
Reviewed by: Pia Giovannelli
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013).
In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is “Liver mitochondria isolation” in which the liver is dissected from mice to obtain mitochondria. “Mitochondria labeling with JC-1 and size fractionation” is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the “Real-time MOMP measurements” protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).
Keywords: Apoptosis Mitochondria BCL-2 Family MOMP Mitochondrial Fractionation
Materials and Reagents
50 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200251 )
Petri dish, 100 x 15 mm (Fisher Scientific, catalog number: FB0875712 )
15 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200250 )
1.5 ml Micro centrifuge tube (USA Scientific, catalog number: 1615-5510 )
Gravity chromatography column (Thermo Fisher Scientific, PierceTM, catalog number: 29920 )
Pasteur pipet (Santa Cruz Biotechnology, catalog number: sc-204537 )
96-well plate, flat bottom, black polystyrene (Corning, CostarTM, catalog number: 3915 )
C57BL/6 mice (Charles River Laboratories, catalog number: 027 )
1x phosphate buffered saline (PBS), pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) (Fisher Scientific, catalog number: BP24384 )
Trehalose (Sigma-Aldrich, catalog number: 1673715 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
[4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid]-KOH (HEPES) (Sigma-Aldrich, catalog number: H0527 )
KCl (Sigma-Aldrich, catalog number: P9541 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Ethyleneglycoltetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
Bovine serum albumin-fraction V (Sigma-Aldrich, catalog number: A9418 )
Protease inhibitor cocktail (Thermo Fisher Scientific, HALTTM, catalog number: 78430 )
Sepharose CL-2B resin (Sigma-Aldrich, catalog number: CL2B300 )
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Thermo Fisher Scientific, catalog number: T3168 )
Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 )
Recombinant BAX [purified using the IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system (New England Biolabs, catalog number: E6901S )]
β-octylglucoside (OG) (Sigma-Aldrich, catalog number: O8001 )
Dimethylsulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100 )
Magnesium Chloride Hexahydrate (Fisher Scientific, catalog number: M33-500 )
Trehalose isolation buffer (TIB) (see Recipes)
JC-1 loading buffer (see Recipes)
Liposome buffer (see Recipes)
Equipment
Dissection tools (scissors, scalpel, forceps)
Razor blade (Daigger Scientific, catalog number: EF7281A )
15 ml Potter-Elvehjem dounce homogenizer (Omni International, catalog number: 07-358044 )
Swing bucket centrifuge (Thermo Fisher Scientific, Thermo ScientificTM SorvallTM, model: Legend XTR )
Spectrophotometer (Fisher Scientific, GE Healthcare UltrospecTM, model: Ultrospec 7000 )
Water bath (Fisher Scientific, IsoTempTM, model: 215 )
Tabletop centrifuge (Thermo Fisher Scientific, SorvallTM LegendTM, model: Micro 21 )
Fluorescence plate reader (Biotek, model: Synergy H1 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Renault, T. T., Luna-Vargas, M. P. and Chipuk, J. E. (2016). Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement. Bio-protocol 6(15): e1892. DOI: 10.21769/BioProtoc.1892.
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Cell Biology > Organelle isolation > Mitochondria
Cancer Biology > Cell death > Animal models
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1,893 | https://bio-protocol.org/exchange/protocoldetail?id=1893&type=0 | # Bio-Protocol Content
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Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR
Naveen Kumar Kalagatur
ND Nirmaladevi Dhamodaran
Chandranayaka Siddaiah
Venkataramana Mudili
MS Murali Harishchandra Sreepathi
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1893 Views: 9339
Edited by: Arsalan Daudi
Reviewed by: Arsheed Hussain SheikhPriyanka Das
Original Research Article:
The authors used this protocol in Oct 2015
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Oct 2015
Abstract
Fusarium graminearum has been given special attention in the context of agricultural commodities due to its ability to grow in diverse climatic conditions, and to produce different mycotoxins including zearalenone (ZEA) and type-B trichothecenes, which cause ill health effects on humans, animals and plants. The application of synthetic antifungal agents for the control of F. graminearum result in negative health impacts in livestock and humans and the upsurge of resistant organisms as well. Therefore, there is a need to propose proper food grain management practices, including the application of herbal antifungal and mycotoxin controlling agents, to reduce the growth of toxigenic F. graminearum as well as the production of ZEA in agricultural commodities. Ocimum sanctum also known as Holy Basil or Tulsi is widely used as a medicinal plant in Ayurveda. The current protocol demonstrates to quantify the antifungal activity of O. sanctum L. essential oil (OSEO) as reflected by the decreased F. graminearum growth and ZEA production. Antifungal activities of OSEO are carried out by micro well dilution method and further validated quantitatively by scanning electron microscopic methods. Effects of OSEO on ZEA production is analysed by Quantitative reverse transcription PCR (RT-qPCR) and Ultra high performance liquid chromatography (UHPLC) methods from a broth culture of F. graminearum. Anti-mycotoxic efficacy of OSEO is assessed directly on F. graminearum inoculated maize grains. The protocol efficiently assessed the activity of OSEO as an herbal antagonistic agent against fungal infestation and ZEA production by F. graminearum. The protocol can be used to test a wide variety of herbal compounds for antifungal activity against F. graminearum or with modifications on other mycotoxigenic fungi, an important intervention in food safety and processing industries where the fungal infestation is a major concern.
Keywords: Zearalenone Fusarium Ocimum sanctum oil UHPLC Q-RTPCR
Materials and Reagents
0.22 µm Millex-GP syringe filter unit (Sigma-Aldrich, catalog number: Z359904 )
96-well microtiter plates (Eppendorf, catalog number: 0030602200 )
Column C18, 5 µm, 250 x 4.6 mm (Phenomenex, catalog number: 00G-4041-E0 )
Carbon Conductive Tape (Ted Pella, Inc., catalog number: 16084-7 )
Glass slides (HiMedia, catalog number: CG081 )
Whatman No.1 paper (Sigma-Aldrich, catalog number: Z274852 )
Zearalenone producing F. graminearum [The Microbial Type Culture Collection and Gene Bank, (MTCC), catalog number: 1893 ]
Maize grains (Local agricultural market, Mysore, India)
Zearalenone standard (Sigma-Aldrich, catalog number: Z2125 )
Dimethyl sulfoxide (Merck Millipore, catalog number: 317275 )
Distilled water
Sodium chloride (NaCl) (Merck Millipore, catalog number: 1064040500 )
Potassium chloride (KCl) (Merck Millipore, catalog number: 1049360250 )
Sodium phosphate dibasic (Na2HPO4) (Merck Millipore, catalog number: 567550-1KG )
Potassium phosphate monobasic (KH2PO4) (Merck Millipore, catalog number: 1048730250 )
0.1 M sodium cacodylate buffer, pH 6.5 (Sigma-Aldrich, catalog number: 70114 )
25% glutaraldehyde (Merck Millipore, catalog number: 354400 )
Acetonitrile (Merck Millipore, catalog number: 100030 )
Ethanol (Merck Millipore, catalog number: 100983 )
Gold foil (Sigma-Aldrich, catalog number: 265829 )
Immunoaffinity column of ZEA (Vicam, catalog number: G1026 )
iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad Laboratories, catalog number: 1708892 )
Liquid nitrogen (Local suppliers, Mysore, India)
Nuclease-free water (Qiagen, catalog number: 129114 )
Nystatin (Sigma-Aldrich, catalog number: N6261 )
Ocimum sanctum L. essential oil (OSEO) (prepared as describe in Procedure step 2)
Peptone (HiMedia, catalog number: RM001-500G )
Porcelain mortar (Sigma-Aldrich, catalog number: Z529508 )
RNA easy plant Mini kit (Qiagen, catalog number: 74903 )
Sabouraud dextrose agar (HiMedia, catalog number: M063-500G )
Sabouraud dextrose broth (HiMedia, catalog number: M033-500G )
Synthesized primer sequences (Sigma-Aldrich, Bangalore, India)
Tween 80 (Merck Millipore, catalog number: 822187 )
Lactophenol-cotton blue (HiMedia, catalog number: S016-500ML )
Phosphate-buffered saline (pH 7.4) (see Recipes)
Lactophenol-cotton blue staining solution (see Recipes)
Equipment
Milli-Q integral water purification system (Merck Millipore, catalog number: ZRXQ005WW )
Aluminum stubs (Ted Pella, Inc., catalog number: 16111N )
Autoclave (Medica Instrument Manufacturing Company, model: 7431PAD )
Microcentrifuge (Sigma-Aldrich, Eppendorf, model: 5415 R )
Centrifuge (Eppendorf, model: 5430 R )
Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
Hot-air oven (Memmert, model: UFP800DW )
Incubator (Bio-age, model: BSR-R2 )
Real-Time PCR system (Roche Diagnostics, Light cycler®, model: 480 )
0.5-10 µl micropipette (Eppendorf Research plus, catalog number: 3120000020 )
20-200 µl micropipette (Eppendorf Research plus, catalog number: 3120000054 )
100-1,000 µl micropipette (Eppendorf Research plus, catalog number: 3120000062 )
Microscope (Leica Microsystems, model: Leica DM 1000 LED )
250 ml Erlenmeyer flasks (Duran Group, catalog number: 21 216 36 )
500 ml Erlenmeyer flasks (Duran Group, catalog number: 21 216 44 )
NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, catalog number: ND-8000-GL )
Nexera UHPLC system (Shimadzu Corporation, model: Nexera X2 )
Scanning electron microscope (FEI, model: Quanta 200 )
Note: This product has been discontinued by the manufacturer [Replaceable items (FEI, model: Quanta 250/450/650 )].
Shaker Incubator (Bio-age, model: BSR-R1 )
Sputter coater (Quorum Technologies, model: SC7620 )
Water bath (NUVE, model: NB 5 )
Weighing balance (Denver instruments, model: TB-215D )
Software
GeneRunner software version 5.0.47 Beta
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kalagatur, N. K., Dhamodaran, N., Siddaiah, C., Mudili, V. and Sreepathi, M. H. (2016). Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR. Bio-protocol 6(15): e1893. DOI: 10.21769/BioProtoc.1893.
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Category
Microbiology > Antimicrobial assay > Antifungal assay
Microbiology > Microbe-host interactions > Fungus
Plant Science > Plant biochemistry > Lipid
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1,894 | https://bio-protocol.org/exchange/protocoldetail?id=1894&type=0 | # Bio-Protocol Content
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Peer-reviewed
Capillary Electrophoresis in Hydroxyethylcellulose Solutions for the Analysis of dsDNA, dsRNA, and siRNA
CL Chenchen Liu
YY Yoshinori Yamaguchi
XD Xiaoming Dou
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1894 Views: 8964
Edited by: Samik Bhattacharya
Reviewed by: Amit Dey
Original Research Article:
The authors used this protocol in Jul 2015
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Abstract
Capillary electrophoresis (CE) is identified as a promising technology for the study of nucleic acid molecules because of its high efficiency, high throughput with automation and integration. Compared to the traditional method of slab gel electrophoresis (SGE), the advantages of CE cannot be emphasized more. Most of CE process, including sample injection, detection and data analysis, is able to be automated which will save great labor for industrial and research labs. CE used the separation channel with micrometer-scale diameter, so the joule heat is easy to be dissipated during electrophoresis. Thus high separation voltage (> 100 V/cm) is allowed in CE while in SGE (usually ~10 V/cm) it usually causes severe band broadening. Because the band broadening is restrained efficiently in CE, it is capable of detecting minute samples and becoming more sensitive than SGE. The advantage of allowing high voltage consequently speeds up the CE separation and yields a better throughput compared to SGE. CE costs less reagents, for example buffer solutions, sieving matrix, dye reagents etc. In addition, the micrometer-scale channel is easy to be integrated with upstream and downstream sample treatment units, forming a lab on a chip. This merit of CE already attracted considerable interests among researchers from various areas.
The difficulties of CE involve filling the gels (agarose or cross-linked polyacrylamide) into the capillary tube. Also, the reproducibility and the life-time of the gel-capillary are limited. But the small-diameter capillary allows to use replaceable polymer solutions, which can efficiently prevent the convection of the separation buffer. Polymer solutions are easier to be filled into the capillary and yield more stable separations. Thus, those difficulties are resolved by doing capillary polymer electrophoresis (CPE), which is going to be described in this protocol.
Several separation modes, for example, capillary gel electrophoresis (CGE), CPE, capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and so on, have been developed for analysis of different kinds of molecules. Here, we introduce the protocol for CPE in detail, which is for the separation of dsDNA, dsRNA (including siRNA) molecules. Polymer solutions are filled into the capillary as a sieving matrix for double strand nucleic acids separation. Hydroxyethylcellulose (HEC) polymer is employed as the sieving polymer in this case. A home-built CE system is described in detail.
Materials and Reagents
Micro-centrifuge or PCR tube (200 μl)
Pipettes (0.1-10 μl and 10-200 μl) and the corresponding tips
0.22 μm filter membrane
dsDNA analytes (Takara, catalog number: 3420A )
dsRNA (including siRNA) analytes (Takara, catalog number: 3430 )
Ultra-pure water, with resistance of 18.25 Ω
2-hydroxyethyl cellulose (MW: 1,300k) (Sigma-Aldrich, catalog number: 434981 )
TBE (Tris-borate-EDTA) powder (Takara, catalog number: T905 )
SYBR Green II RNA Gel Stain, 10,000x concentrate in DMSO (Thermo Fisher Scientific, catalog number: S-7564 )
Storage 1% HEC polymer solution (see Recipes)
0.5x TBE solution (see Recipes)
100x SYBR Green II (see Recipes)
Equipment
Home-built CE system (Figure 1) which includes:
A coated fused silica capillary with a detection window (Polymicro, catalog number: TSP075375 )
A high voltage power supplier (see Note 8)
A fluorescence microscope assembled with a fluorescent excitation equipment (Equipment 1d) and a fluorescent emission collection equipment (Equipment 1e) (Olympus, model: IX73 )
The fluorescent excitation equipment constituted by a mercury lamp and an optical cube, which was used to transmit light from the mercury lamp into 460-495 nm fluorescence excitation spectrum (Olympus, model: U-MWIB3 )
The fluorescent emission collection equipment constituted by an optical filter and a photomultiplier tube (PMT) (Hamamatsu Photonics, catalog number: H8249-101 , for alternate H7827-001)
Note: The optical filter transmits the emission spectrum from dye-nucleonic acid conjugate to the PMT and filters background light from the detection window.
The home-built CE system including a Labview system (National Instrument, model: NI USB 6212 ) and a computer
Note: Users are able to control the high voltage supplier, monitor fluorescent signal from the detection window and collect data from PMT by the LabVIEW software.
Vacuum pump (see Note 9)
Centrifuge (> 3,200 x g)
Ultra-pure water system (see Note 10)
Figure 1. Schematic diagram of the assembly of the CE system
Software
LabVIEW software
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Liu, C., Yamaguchi, Y. and Dou, X. (2016). Capillary Electrophoresis in Hydroxyethylcellulose Solutions for the Analysis of dsDNA, dsRNA, and siRNA. Bio-protocol 6(15): e1894. DOI: 10.21769/BioProtoc.1894.
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Category
Molecular Biology > DNA > Electrophoresis
Molecular Biology > RNA > Electrophoresis
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1,895 | https://bio-protocol.org/exchange/protocoldetail?id=1895&type=0 | # Bio-Protocol Content
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Bone Marrow Mesenchymal Stem Cells Adhesion Assay
ZY Zhigang Yang
RX Ran Xiao
Published: Vol 6, Iss 15, Aug 5, 2016
DOI: 10.21769/BioProtoc.1895 Views: 10687
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
Mesenchymal stem cells (MSCs) are widespread in adult organisms and involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. As cell adhesion play important roles in cell interactions and signaling, to thoroughly evaluate the adhesion ability of MSCs is of vital importance to clarify the mechanism of self-renewal, proliferation, activation and migration of MSCs in different microenvironments. Based on the method by Siler et al., 2000, we revised the protocol in order to provide details on how to evaluate the adhesion ability of MSCs from bone marrow (BMSCs) on extracellular matrix (ECM) protein laminins. The current protocol can also be easily translated to MSCs with other treatments or ECMs such as collagens, fibronectin, etc.
Keywords: Mesenchymal stem cells Extracellular matrix Adhesion Stem cells Laminin
Materials and Reagents
96 well culture plate (Sigma-Aldrich, Corning® Costar®, catalog number: CLS3595 )
Human bone marrow derived MSCs, isolated and cultured as previously described (Kern et al., 2006)
Note: Cells in the third cell passage were used.
Laminin 511&521 (Biolamina, catalog number: LN511 ; LN521 )
DMEM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11965092 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10099141 )
Trypsin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25200-056 )
Soybean trypsin inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17075-029 )
BSA (Sigma-Aldrich, catalog number: V900933 )
Blocking buffer (0.5% BSA in PBS without Ca2+ and Mg2+)
Wash buffer (0.1% BSA in PBS without Ca2+ and Mg2+)
2% SDS (Sigma-Aldrich, catalog number: 71729 )
4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
KCl (Sigma-Aldrich, catalog number: 746436 )
Na2HPO4 (Sigma-Aldrich, catalog number: 795410 )
KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
CaCl2·2H2O (Sigma-Aldrich, catalog number: 223506 )
MgCl2·6H2O (Sigma-Aldrich, catalog number: M9272 )
HCl (Sinopharm chemical reagent Being Co.ltd, catalog number: 10011008 )
Crystal violet powder (Sigma-Aldrich, catalog number: C6158 )
Ethanol (Sinopharm chemical reagent Being Co.ltd, catalog number: 10009159 )
PBS with/without Ca2+ and Mg2+ (see Recipes)
Note: Commercial DPBS with/without calcium and magnesium can also be used.
0.1% crystal violet staining solution (see Recipes)
Equipment
Small shaker for microtiter plate (IKA, model: MS3 Digital )
Scanner (UMAX, model: POWERLOOK 2100XL )
Series II 3110 Water-Jacketed CO2 chamber (Thermo Fisher Scientific, FormaTM, catalog number: 3111 )
Microscope (Nikon, model: ECLIPSE TE2000-S )
Microplate reader (ThermoFisher Scientific, model: Multiscan MK3 ) or spectrometer with 550 nm wavelength available.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yang, Z. and Xiao, R. (2016). Bone Marrow Mesenchymal Stem Cells Adhesion Assay. Bio-protocol 6(15): e1895. DOI: 10.21769/BioProtoc.1895.
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Category
Stem Cell > Adult stem cell > Mesenchymal stem cell
Cell Biology > Cell movement > Cell motility
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1,896 | https://bio-protocol.org/exchange/protocoldetail?id=1896&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.
Agnès Groisillier
Thierry Tonon
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1896 Views: 8295
Edited by: Valentine V Trotter
Reviewed by: Yanjie Li
Original Research Article:
The authors used this protocol in Feb 2014
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Feb 2014
Abstract
Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of a recombinant M1Pase of Ectocarpus sp. The M1Pase enzyme catalyzes the conversion of mannitol-1-phosphate to mannitol (Figure 1).
Figure 1. Reaction catalyzed by a mannitol-1-phosphatase
Keywords: Mannitol cycle Mannitol-1-phosphatase Ectocarpus sp. Brown algae
Materials and Reagents
UV-Star® PS microplate (96 well) (Greiner Bio-One GmbH, catalog number: 655801 )
0.22 µm filter
Escherichia coli BL21 (DE3)
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Purified recombinant His-tagged M1Pase
Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ M1Pase vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare) onto an Äkta avant system (GE Healthcare). The complete purification protocol is described in details in Groisillier et al. (2014).
MgCl2 (Sigma-Aldrich, catalog number: M8266 )
MilliQ water
Malachite green phosphate assay kit (Gentaur, catalog number: POMG-25H )
Note: Malachite green phosphate assay protocol is available at https://www.bioassaysys.com/Datasheet/POMG.pdf.
Examples of chemicals to be tested to assess substrate specificity
D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
D-fructose-1-phosphate (Sigma-Aldrich, catalog number: F1127 )
D-glucose-1-phosphate (Sigma-Aldrich, catalog number: G9380 )
D-mannose-6-phosphate (Sigma-Aldrich, catalog number: M3655 )
D-glucose-6-phosphate (Sigma-Aldrich, catalog number: G7879 )
D-fructose-6-phosphate (Sigma-Aldrich, catalog number: F3627 )
NaCl (Sigma-Aldrich, catalog number: 71380 )
1 M Tris-HCl (pH 7.5) (see Recipes)
100 mM MgCl2 (see Recipes)
5 M NaCl (see Recipes)
Equipment
NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 2000 )
Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
Software
Hyper32 (Informer Technologies, http://hyper32.software.informer.com/)
Microsoft Excel
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Groisillier, A. and Tonon, T. (2016). Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.. Bio-protocol 6(16): e1896. DOI: 10.21769/BioProtoc.1896.
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Category
Plant Science > Phycology > Protein
Biochemistry > Protein > Activity
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1,897 | https://bio-protocol.org/exchange/protocoldetail?id=1897&type=0 | # Bio-Protocol Content
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15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice
YL Yongqiang Liu
BH Bin Hu
Chengcai Chu
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1897 Views: 11629
Edited by: Tie Liu
Reviewed by: Pooja Saxena
Original Research Article:
The authors used this protocol in Jun 2015
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Jun 2015
Abstract
15N is a nonradioactive heavy isotope of nitrogen, widely used for biochemical and physiological research in plants. For instance, 15N-KNO3 was used as the nitrogen source in plants in order to investigate nitrate uptake activity and transport from roots to shoots (Lin et al., 2008). Here, we describe a detailed pipeline used for labeling living rice (Oryza sativa) plants with 15N-KNO3 and determination of net nitrate uptake and transport activity, and this protocol was proved to be valid in Arabidopsis and rice (Lin et al., 2008; Hu et al., 2015).
Keywords: Nitrate uptake Nitrate transport Rice Isotope
Materials and Reagents
96-well plate
Rice seeds (Zhonghua11, ZH11)
NaClO [Sinopharm Chemical Reagent Co.,Ltd (SCRC), catalog number: 7681-52-9 ]
KNO3 (SCRC, catalog number: 7757-79-1 )
K15NO3 (Sigma-Aldrich, catalog number: 57654-83-8 )
CaCl2 (SCRC, catalog number: 10043-52-4 )
MgSO4·7H2O (SCRC, catalog number: 10034-99-8 )
KH2PO4 (SCRC, catalog number: 7778-77-0 )
FeSO4·7H2O (SCRC, catalog number: 7782-63-0 )
EDTA-Na2 (SCRC, catalog number: 6381-92-6 )
NaSiO3·9H2O (SCRC, catalog number: 13517-24-3 )
H3BO3 (SCRC, catalog number: 10043-35-3 )
CuSO4·5H2O (SCRC, catalog number: 7758-99-8 )
ZnSO4·7H2O (SCRC, catalog number: 7446-20-0 )
MnCl2·4H2O (SCRC, catalog number: 13446-34-9 )
Na2MoO4·2H2O (SCRC, catalog number: 10102-40-6 )
CaSO4·2H2O (SCRC, catalog number: 10101-41-4 )
Modified Kimura B solution (see Recipes)
5 mM 15N-KNO3 (see Recipes)
0.1 mM CaSO4 solution (see Recipes)
Equipment
Growth chamber (SANYO, model: MLR-351H )
Isotope ratio mass spectrometer(Thermo Fisher Scientific, model: Finnigan Delta Plus XP) with elemental analyzer (Thermo Fisher Scientific, model: Flash EA 1112 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Liu, Y., Hu, B. and Chu, C. (2016). 15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice. Bio-protocol 6(16): e1897. DOI: 10.21769/BioProtoc.1897.
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Category
Plant Science > Plant physiology > Nutrition
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1,898 | https://bio-protocol.org/exchange/protocoldetail?id=1898&type=0 | # Bio-Protocol Content
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Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin
HH Hiroyuki Hirai
TF Takehito Furukawa
YK Yuya Katsuragi
FC Fang-Sik Che
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1898 Views: 8559
Edited by: Zhaohui Liu
Reviewed by: Chong HeArsalan Daudi
Original Research Article:
The authors used this protocol in Jun 2015
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Jun 2015
Abstract
Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease characterized by the deposition of brown stripes on the sheaths of infected plants. We previously reported that flagellin isolated from the rice avirulent A. avenae N1141 strain induces PTI, while flagellin isolated from the rice virulent A. avenae K1 strain does not induce PTI. To examine the molecular mechanism of specific PTI induction by N1141 flagellin, highly purified flagellin from N1141 or K1 strains is required. Here, we describe a high quality purification method for the A. avenae flagellins and for using it in PTI induction study.
Keywords: Flagellin Acidovorax avenae Purification Plant
Materials and Reagents
0.22 µm sterilization filter (Merck Millipore, catalog number: SLGS033SB )
Nitrocellulose membrane (GE Healthcare, code number: 10401196 )
Collodion-coated grid (Ted Pella, Inc., catalog number: 12575-CU )
Cultured rice cells of line OC (Oryza sativa C5928; obtained from RIKEN BioResource Center)
Acidovorax avenae N1141 (MAFF 301141) and K1 (MAFF301755) (National Institute of Agrobaiological Sciences genebank, catalog number: MAFF 301141 ; MAFF301755 )
Skim milk powder (Wako, catalog number: 190-12865 )
Sodium hydrogen L(+)-glutamate monohydrate (Wako, catalog number: 198-02035 )
Luria-Bertani (LB) liquid medium (MO BIO laboratories, catalog number: 12107-05 )
2-amino-2-hydroxymethyl-1,3-propanediol (Tris) (Wako, catalog number: 204-07885 )
NaCl (Nacalai Tesque, catalog number: 31320-05 )
KCl (Nacalai Tesque, catalog number: 28514-75 )
1% (w/v) phosphotungstic acid (pH 6.9) (Sigma-Aldrich, catalog number: 79690 )
Sodium dodecyl sulfate (SDS) (Wako, catalog number: 191-07145 )
Molecular-weight marker low (APRO, catalog number: SP-0110 )
Anti-flagellin rabbit antibody (Eurofins Genomics)
Note: Flagellin purified from A. avenae N1141 strain as antigen was injected in rabbit. Anti-flagellin rabbit antibody was purified from rabbit antiserum (anti-flagellin) using flagellin purified from A. avenae N1141 strain.
Goat HRP conjugated anti-rabbit IgG antibody (H + L chain) [Medical & Biological Laboratories (MBL), catalog number: 458 ]
ECL plus Western blotting detection reagents (GE Healthcare, code number: RPN2132 )
Hybri-Bag (Cosmo Bio, catalog number: S-1001 )
Potassium ferricyanide (Wako, catalog number: 169-03721 )
KNO3 (Nacalai Tesque, catalog number: 28704-85 )
(NH4)2SO4 (Wako, catalog number: 019-03435 )
MgSO4·7H2O (Nacalai Tesque, catalog number: 21003-75 )
CaCl2·2H2O (Nacalai Tesque, catalog number: 06731-05 )
NaH2PO4·2H2O (Wako, catalog number: 192-02815 )
MnSO4·5H2O (Nacalai Tesque, catalog number: 21229-35 )
ZnSO4·7H2O (Nacalai Tesque, catalog number: 37011-75 )
CuSO4·5H2O (Nacalai Tesque, catalog number: 09605-04 )
H3BO3 (Wako, catalog number: 021-02195 )
Na2MoO4·2H2O (Nacalai Tesque, catalog number: 31621-52 )
EDTA·2Na (DOJINDO, catalog number: 345-01865 )
FeSO4·7H2O (Wako, catalog number: 098-01085 )
MS vitamin powder 1,000x (Sigma-Aldrich, catalog number: M7150-100ML )
2, 4-dichlorophenoxyacetic acid (Wako, catalog number: 040-18532 )
Sucrose (Wako, catalog number: 196-00015 )
3-mercapto-1,2-propanediol (Wako, catalog number: 131-16451 )
Glycerol (Nacalai Tesque, catalog number: 17018-25 )
Bromophenol blue (BPB) (Wako, catalog number: 021-02911 )
Glycine (Wako, catalog number: 077-00735 )
Coomassie brilliant blue R-250 (CBB) (Wako, catalog number: 031-17922 )
Methanol (Wako, catalog number: 137-01823 )
Acetic acid (Wako, catalog number: 017-00251 )
Polyoxyethylene sorbitan monolaurate (Tween 20) (Wako, catalog number: 167-11515 )
KH2PO4 (Wako, catalog number: 169-04245 )
Skimmed milk solution (see Recipes)
Acidovorax avenae N1141 and K1 strain skimmed milk stock solution (see Recipes)
25 mM TBS buffer (pH 7.4) (see Recipes)
20x R2 Major solution (see Recipes)
1,000x R2 Minor solution (see Recipes)
500x Fe liquid solution (see Recipes)
1,000x MS vitamin solution (see Recipes)
2, 4-dichlorophenoxyacetic acid solution (see Recipes)
R2S medium (pH 5.6) (see Recipes)
2x sample buffer (pH 6.8) (see Recipes)
Electrophoresis buffer (see Recipes)
CBB staining solution (see Recipes)
Destaining solution (see Recipes)
Transfer buffer (see Recipes)
Tris buffered saline with Tween 20 (TBST) buffer (see Recipes)
Blocking buffer (see Recipes)
50 mM potassium phosphate buffer (pH 7.9) (see Recipes)
Luminol solution (see Recipes)
Potassium ferricyanide solution (see Recipes)
Equipment
Rotary shaker (TAITEC, model: NR-20 )
High-speed refrigerated micro centrifuge (TOMY, model: MX-300 )
High-speed refrigerated centrifuge (Hitachi koki, model: CR20G )
Ultracentrifuge (Hitachi koki, model: CP70MX )
Fiber blender (Panasonic, model: MX-X58-SW )
Incubator (TAITEC, model: BR-42FL· MR )
Transmission electron microscope (Hitachi, model: H-7100 )
Plant growth chamber (NK system, model: LH-411SP )
Electrophoresis tank (ATTO, model: AE-6530M )
Semi-dry blotter (Bio-Rad, catalog number: 1703940JA )
Luminescent image analyzer (GE Healthcare, model: ImageQuant LAS 4000 )
Lumi-counter (ATTO, model: AB-2350 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hirai, H., Furukawa, T., Katsuragi, Y. and Che, F. (2016). Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin. Bio-protocol 6(16): e1898. DOI: 10.21769/BioProtoc.1898.
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Category
Biochemistry > Protein > Immunodetection
Plant Science > Plant immunity > Disease bioassay
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1,899 | https://bio-protocol.org/exchange/protocoldetail?id=1899&type=0 | # Bio-Protocol Content
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Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
Felipe Vences-Catalán
SL Shoshana Levy
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1899 Views: 10265
Edited by: Lee-Hwa Tai
Reviewed by: Clara Lubeseder-MartellatoToshitsugu Fujita
Original Research Article:
The authors used this protocol in Nov 2015
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Abstract
Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.
Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4+CD25+ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4+ T cells are enriched by negative selection, followed by positive selection of CD25+ T cells. Tumor-induced purified Tregs (CD3+CD4+CD25+FoxP3+) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.
Keywords: Tregs Adoptive transfer Tumor Immune suppression
Materials and Reagents
70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
1 ml syringe (BD, catalog number: 309659 )
30 G x 1/2 needle (BD, catalog number: 305106 )
LD columns (Miltenyi Biotec, catalog number: 130-042-901 )
MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
15 and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352096 and 352070 )
Mice
Note: We use 6-8 weeks old female Balb/c or C57BL6 mice, but the protocol is applicable to any mouse strain as long as both donor and recipient mice are from the same genetic background.
Cells
Note: We use breast cancer cell lines 4T1 or E0771 syngeneic to Balb/c or C57Bl6 respectively.
Fetal calf serum (FCS) (GE life sciences, HyCloneTM, FetalClone®III, catalog numer: SH30109.03 )
Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-22 )
Note: 5 ml of this solution was used in 500 ml of RPMI 1640 media.
PBS (Corning, Cellgro, catalog number: 21-031-CV )
ACK buffer (Quality Biological, catalog number: 118-156-101 )
CD4+CD25+ Regulatory T cell Isolation kit (Miltenyi Biotec, catalog number: 130-091-041 )
Components of the kit:
1 ml CD4+CD25+ Regulatory T cell Biotin-antibody cocktail
2 ml Anti-biotin Microbeads
1 ml CD25-PE
1 ml Anti-PE Microbeads
RPMI 1640 (Corning, Cellgro, catalog number: 10-040-CV )
Bovine serum albumin (BSA) (Sigma Aldrich, catalog number: A9647-500G )
Note: A solution of 0.5% in PBS was used.
EDTA (Sigma Aldrich, catalog number: E-5134 )
Note: A solution of 2 mM in PBS was used.
Optional: CD3 FITC (clone:145-2C11) (BD Pharmingen)
Optional: CD25 PE (clone:PC61.5) (BD Pharmingen)
Optional: CD4 PerCP (clone:H129.19/RM4-5) (BD Pharmingen)
Optional: FoxP3 APC (clone:FJK16s) (BD Pharmingen)
MACS Buffer (see Recipes)
Equipment
MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
Centrifuge (Eppendorf, model: 5810 R )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Vences-Catalán, F. and Levy, S. (2016). Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs). Bio-protocol 6(16): e1899. DOI: 10.21769/BioProtoc.1899.
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Category
Immunology > Immune cell isolation > Lymphocyte
Cancer Biology > Tumor immunology > Tumor microenvironment
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19 | https://bio-protocol.org/exchange/protocoldetail?id=19&type=1 | # Bio-Protocol Content
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RNA Preparation for Microarray Experiments
Bio-protocol Editor
Published: Jan 20, 2011
DOI: 10.21769/BioProtoc.19 Views: 15886
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Abstract
This protocol describes a simple and relatively general method to extract total RNA from a yeast culture using the Qiagen RNeasy kit. In it, total RNA is treated with DNase I and cleaned up to be suitable for microarray experiments. Therefore, this protocol can be used to generate a large yield of high quality total RNA from yeast cells (S. cerevisiae and S. pombe).
Materials and Reagents
S. pombe cells
Qiagen RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
DNase I (Roche Diagnostics, catalog number: 04716728001 )
RNA STAT based on "S. Pombe RNA Prep"
DEPC water
EDTA
RLT buffer
Equipment
Standard bench-top centrifuge with 1.5 ml Eppendorf tube capacity
Calibrated standard laboratory pipettes
Procedure
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial genetics > RNA
Molecular Biology > RNA > RNA extraction
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190 | https://bio-protocol.org/exchange/protocoldetail?id=190&type=1 | # Bio-Protocol Content
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Transfection of S2 Cell with DNA Using CellFectin Reagent
DC Darran G. Cronshaw
Published: Mar 5, 2012
DOI: 10.21769/BioProtoc.190 Views: 22168
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This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.
Materials and Reagents
Drosophila Schneider 2 (S2) cells (Life Technologies, Invitrogen™, catalog number: R690-07 )
Dros-SFM (Life Technologies, Invitrogen™, catalog number: 10797-025 )
pHASβAG (pRmHA-3 vector with SDF-1β-FLAG inserted. Contains a metallothionein promoter inducible with Cu2+)
pUC-Hsneo (contains the neomycin-resistant cassette required for stable selection)
CellFectin reagent (Life Technologies, Invitrogen™, catalog number: 10362-010 )
FBS (Life Technologies, Invitrogen™, catalog number: 10437-028 )
Penicillin-Streptomycin (P/S) (Life Technologies, Invitrogen™, catalog number: 15140-122 )
G418 sulphate (Life Technologies, Invitrogen™, catalog number: 11811-031 )
CuSO4 (Sigma Aldrich, catalog number: C8027 )
HT supplement (Life Technologies, Invitrogen™, catalog number: 11067-030 )
Transfection solutions (see Recipes)
Equipment
6-well plate (Corning Incorporated, catalog number: 353046 )
24-well plate (Corning Incorporated, catalog number: 353047 )
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Molecular Biology > DNA > Transformation
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1,900 | https://bio-protocol.org/exchange/protocoldetail?id=1900&type=0 | # Bio-Protocol Content
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Macrophage Polarization by Tumor-induced MDSCs Assay
Felipe Vences-Catalán
Minu K. Srivastava
SL Shoshana Levy
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1900 Views: 12967
Edited by: Lee-Hwa Tai
Reviewed by: Clara Lubeseder-MartellatoToshitsugu Fujita
Original Research Article:
The authors used this protocol in Nov 2015
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Nov 2015
Abstract
Myeloid derived suppressor cells (MDSCs) are a subset of granulocytes (immature myeloid cells) that exploit a variety of mechanism to modulate the innate and adaptive immune system. MDSCs are present normally in the body, but their numbers increase during inflammation and in cancer, promoting an immunosuppressive microenvironment. In addition to MDSCs, macrophages also play an important role during cancer development. There are two subsets of tumor associated macrophages (TAMs): M1 and M2. M1 are “anti-tumor” macrophages that are activated by interferon gamma (IFN-γ) and/or Lipopolysaccharide (LPS) and secrete high amount of interleukin 12 (IL-12) thereby inducing a Th1 anti-tumor immune response. M2 or “pro-tumorigenic” macrophages are activated by interleukin 4 (IL-4) and interleukin 10 (IL-10) and secrete large amounts of IL-10, which promotes tumor progression (Gabrilovich et al., 2012).
Interaction between MDSCs and macrophages in the tumor microenvironment was shown to enhance immune suppression mediated by these subsets. MDSCs influence TAMs by producing IL-10 that, in turn, induces a down-regulation of IL-12 and polarizes M1 into M2 macrophages. In our study, we use the following protocol to evaluate the ability of tumor induced MDSCs to polarize LPS activated M1 into M2 macrophages (Vences-Catalan et al., 2015). This protocol was adapted from a previous study (Sinha et al., 2007).
Keywords: MDSCs Macrophage Tumor Macrophage polarization Cytokines
Materials and Reagents
70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
1 and 10 ml syringe (BD, catalog number: 309659 and 309604 )
15 and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352096 and 352070 )
18 gauze needle (BD, catalog number: 305196 )
24-well plates (Corning, Falcon®, catalog number: 351147 )
Mice
Note: We use 6-8 weeks old female Balb/c mice, but any strain of mice can be used as long as both macrophages and MDSCs are from the same genetic background.
Cells
Note: We use 4T1 breast cancer cell line syngeneic to Balb/c; this tumor model is known to induce a strong accumulation of MDSCs in blood, spleen and tumor.
Thioglycolate (BD, catalog number: 211716 )
Note: A 3% solution in water and sterilized in autoclave at 121 °C for 15 min has been used.
PBS (Corning, Cellgro, catalog number: 21-031-CV )
RPMI 1640 (Corning, Cellgro, catalog number: 10-040-CV )
DMEM media (Corning, Cellgro, catalog number: 10-017-CV )
Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-22 )
Note: 5 ml of this solution was used in 500 ml of RPMI 1640 media.
Fetal calf serum (FCS) (GE Life Sciences, HyCloneTM, FetalClone®III, catalog number: SH30109.03 )
ACK buffer (Quality Biological, catalog number: 118-156-101 )
IL-12p70 and IL-10 ELISA kit (Biolegend, Legend MaxTM, catalog number: 431417 and 433607 )
CD11b PE (clone: M1/70) (BD, catalog number: 553311 )
F4/80 APC (clone: BM8) (eBioscience, catalog number: 17-4801-82 )
Anti Ly6G and Ly6c (Gr1) APC (clone: RB6-8C5) (BD, catalog number: 553129 )
LPS 1 mg/ml (Sigma-Aldrich, catalog number: L3012-5MG )
Note: If using a different tumor model where MDSCs in blood or spleen represents a small percentage of total Peripheral Blood Mononuclear Cells or splenocytes, respectively, purify MDSCs with myeloid-derived suppressor cell isolation kit and follow instructions according to manufacturers protocol (Miltenyi Biotec, catalog number: 130-094-538 ).
Equipment
Centrifuge (Eppendorf, model: 5810 R )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Myeloid derived suppressor cells
Cancer Biology > Tumor immunology > Tumor microenvironment
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Could you tell me if the IL-4 used in the experiment was human or mouse?
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1,901 | https://bio-protocol.org/exchange/protocoldetail?id=1901&type=0 | # Bio-Protocol Content
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A Controlled Cortical Impact Mouse Model for Mild Traumatic Brain Injury
Heidi Y Febinger
Hannah E Thomasy
CG Carmelina Gemma
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1901 Views: 12993
Edited by: Soyun Kim
Reviewed by: Xiaoyu LiuXi Feng
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
Traumatic brain injury (TBI) affects millions of people worldwide; however, the immediate impact of TBI and the secondary injury mechanisms are still not fully understood. TBI can cause devastating neuromotor deficits in both acute and chronic stages. Time course studies utilizing animal models of focal TBI have provided essential insight into TBI neuropathology. Here, we describe a surgical technique for creating a mouse model of focal, mild TBI (Dixon et al., 1991; Smith et al., 1995; Bolkvadze and Pitkanen, 2012). Furthermore, we provide protocols for validating TBI models using behavioral tests that examine post-traumatic neuromotor deficits resulting from TBI neuropathology (Fujimoto et al., 2004; Febinger et al., 2015; Smith et al., 1995; Bolkvadze and Pitkanen, 2012).
Keywords: TBI Concussion Head trauma
Materials and Reagents
Sterile cotton tipped applicators
Gauze pads (ATC Medical, CurityTM, catalog number: 686132 )
Nolvasan surgical scrub (Zoetis Inc., catalog number: 8NOL413 )
Adult C57BL6/J laboratory mice (6 weeks old)
70% ethanol
Isoflurane
Opthalmic ointment
3.0% hydrogen peroxide solution
Sterile saline
VetBond glue (3M, catalog number: 1469SB )
70% isopropanol
Equipment
Surgical Tools
Scalpel handle (Fine Science Tools, catalog number: 10003-12 )
Sterile scalpel blades (Fine Science Tools, catalog number: 10010-00 )
Dumont forceps (Fine Science Tools, catalog number: 11251-10 )
5.0-mm diameter trephine (Figure 1) (Fine Science Tools, catalog number: 18004-50 )
Pin vise (Figure 1) (ProEdge, catalog number: PRO58111 )
Figure 1. Trephine in pin vise
Hot bead sterilizer (Fine Science Tools, catalog number: 18000-45 )
Warm water recirculator (Kent Scientific, catalog number: TP-700 )
Polystyrene weighing boat
Controlled cortical impact (CCI) device (Impact One Stereotaxic Impactor for CCI) (Leica Biosystems, catalog number: 39463920 )
3.0-mm diameter metal piston (Leica Biosystems , catalog number: 39463920)
Small animal stereotaxic instrument with mouse adapters (Kopf Instruments, model: 900 and 923-B )
Scale
Leather hole punch, sharp (Anytime Tools)
Incandescent lamp (50-75 watt)
Inclined plane device, custom made (Figure 2) (The angle board device is 50 x 60 cm in size and is covered with a vertically grooved rubber mat. The platform was attached to a hinge, allowing the researcher to manually adjust the angle of the board.)
Figure 2. Inclined plane (custom made)
Procedure
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How to cite：Febinger, H. Y., Thomasy, H. E. and Gemma, C. (2016). A Controlled Cortical Impact Mouse Model for Mild Traumatic Brain Injury. Bio-protocol 6(16): e1901. DOI: 10.21769/BioProtoc.1901.
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Category
Neuroscience > Behavioral neuroscience > Animal model
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1,902 | https://bio-protocol.org/exchange/protocoldetail?id=1902&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans
JB Jonathon L. Baker
RF Roberta C. Faustoferri
Robert G. Quivey, Jr
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1902 Views: 13014
Edited by: Valentine V Trotter
Reviewed by: Swetha ReddyRolf Lood
Original Research Article:
The authors used this protocol in Dec 2015
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Dec 2015
Abstract
Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD+) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD+/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD+/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969).
Here, we describe in detail an assay to determine the ratio of free intracellular NAD+ to NADH in Streptococcus mutans. This cycling assay is a modified version of the protocol first described by Bernofsky and Swan (Bernofsky and Swan, 1973), using the extraction buffer described by Frezza et al. (2011), followed by the reduced MTT precipitation described by Gibbon and Larher (Gibon and Larher, 1997). As depicted in Figure 1, alcohol dehydrogenase is used to drive a series of redox reactions utilizing exogenously added ethanol and NAD+ from sample extracts as initial substrates, phenazine ethosulfate (PES) as an electron carrier, and thiazolyl blue tetrazolium bromide (MTT) as a terminal electron acceptor. 6 M NaCl is used to stop the reaction. The reduced MTT (formazan dye) is purple in color and can be quantified by measuring absorbance at 570 nm. This protocol is divided into three steps: A. Preparation of cell pellets of S. mutans; B. Preparation of deproteinated cell extracts containing NADtotal or NADH; C. NAD+/NADH cycling assay. This method has proven robust in measuring the NAD+/NADH ratio in S. mutans under a variety of conditions, and should be applicable to other Gram-positive bacteria.
Figure 1. Flowchart illustrating protocol Procedure parts B-C
Keywords: NADH oxidase Bacteria Streptococci Streptococcus mutans Oxidative stress
Materials and Reagents
Glass test tubes, 18 x 150 mm (VWR International, catalog number: 60825-443 )
50 ml centrifuge tubes (Corning, catalog number: 430829 )
Screw-top microcentrifuge tubes with O-ring, 2.0 ml (Laboratory Product Sales, catalog number: L233072 )
Glass beads, 0.1 mm (BioSpec Products, catalog number: 11079101 )
Centrifugal Filters, 0.5 ml, 10,000 MWCO (Merck Millipore Corporation, Amicon, catalog number: UFC501024 )
Microcentrifuge tubes, 1.7 ml (Laboratory Product Sales, catalog number: L211511 )
Streptococcus mutans bacterial strains of interest
Brain Heart Infusion medium (BHI) (BD, DifcoTM, catalog number: 299070 )
Bacto-agar (BD, DifcoTM, catalog number: 214010 )
Tricine (Sigma-Aldrich, catalog number: T5816 )
Sodium hydroxide (Sigma-Aldrich, catalog number: S5881 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
Sodium chloride (J.T. Baker, catalog number: 3624-01 )
Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M2128 )
Phenazine ethosulfate (PES) (Sigma-Aldrich, catalog number: P4544 )
Alcohol dehydrogenase from Saccharomyces cerevisiae (Adh) (Sigma-Aldrich, catalog number: A3263 )
Ethanol, 200 proof (VWR International, Koptec, catalog number: 64175 )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S8875 )
Sodium carbonate (J.T. Baker, catalog number: 3602-01 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Nicotinamide (Sigma-Aldrich, catalog number: N3376 )
Hydrochloric acid (J.T. Baker, catalog number: 9535 )
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
1 M tricine-NaOH (see Recipes)
40 mM EDTA solution (see Recipes)
6 M NaCl (see Recipes)
0.1 M NaCl (see Recipes)
4.2 mM MTT (see Recipes)
16.6 mM PES (see Recipes)
100 U/ml Adh (see Recipes)
NAD+/NADH master mix (see Recipes)
NAD+/NADH extraction buffer (see Recipes)
2 µM NADH (see Recipes)
Equipment
3 mm (1 µl) inoculation loops (VWR, catalog number: 50807-020 )
CO2 incubator (LABEQUIP) (VWR Scientific, catalog number: 10810-744 )
Erlenmeyer flasks, 250 ml (Pyrex)
Mini Beadbeater 8 (BioSpec Products, model: Mini Beadbeater 8 )
Microcentrifuge (Eppendorf, model: 5424 )
Centrifuge (Fisher Scientific, model: Legend RT) with Thermo swinging bucket rotor (Thermo Fisher Scientific, model: TX-750 )
60 °C water bath
37 °C water bath
Vortexer (Vortex Genie 2) (Scientific Industries, model: G560 )
Dark room
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Baker, J. L., Faustoferri, R. C. and Quivey, Jr, R. G. (2016). A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans . Bio-protocol 6(16): e1902. DOI: 10.21769/BioProtoc.1902.
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Category
Microbiology > Microbial metabolism > Other compound
Microbiology > Microbial biochemistry > Other compound
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1,903 | https://bio-protocol.org/exchange/protocoldetail?id=1903&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
MM Michael J. Munson
IG Ian G. Ganley
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1903 Views: 7810
Edited by: Ralph Bottcher
Reviewed by: Shyam SolankiDiana Búzová
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.
Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system.
Keywords: Phosphatidylinositol-3-phosphate VPS34 PIK3C3 PX domain Fluorescence reporter
Materials and Reagents
ZebaTM spin desalting column (Thermo Fisher Scientific, catalog number: 89891 )
22 x 22 m cover glasses, Menzel Glaser (VWR, catalog number: 631-1336 )
Millex-GS syringe filter unit, 0.22 µm (Merck Millipore, catalog number: SLGSV255F )
Slide-A-Lyzer dialysis cassettes, 10K MWCO 12 ml (Thermo Fisher Scientific, catalog number: 66810 )
Microscope slides, 75 x 25 mm (superfrost) (VWR, catalog number: 48311-600 )
BL-21 bacteria (New England Biolabs, catalog number: C2530H )
U2OS cells (ATCC, catalog number: HTB-96 )
SOC medium (Thermo Fisher Scientific, catalog number: 15544034 )
LB broth, Miller (Merck Millipore, catalog number: 71753 )
LB agar, Miller (Merck Millipore, catalog number: 1102835000 )
Ampicillin
IPTG (Sigma-Aldrich, catalog number: I6758 )
Tris (VWR International, catalog number: 103157P )
Sodium chloride (NaCl) (VWR International, catalog number: 27810.364 )
Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
PMSF (Sigma-Aldrich, catalog number: P7626 )
Benzamidine (Sigma-Aldrich, catalog number: 12072 )
Brij-35 (Merck Millipore Corporation, catalog number: 1.01894.1000 )
EGTA (Sigma-Aldrich, catalog number: E4378 )
Glutathione sepharose 4B (GE Healthcare, catalog number: 17-0756 )
InstantBlue Coomassie stain (Expedeon, catalog number: ISB1L )
Alexa Fluor 488 TFP ester (Thermo Fisher Scientific, catalog number: A37570 )
Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, catalog number: A37572 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
HEPES (ForMedium, catalog number: HEPES10 )
Potassium chloride (KCl) (VWR International, catalog number: 26764.260 )
Magnesium acetate (MgAc) (Sigma-Aldrich, catalog number: M5661 )
Potassium glutamate (Sigma-Aldrich, catalog number: G1501 )
DMEM (Thermo Fisher Scientific, Gibco®, catalog number: 11960-044
PBS pH 7.4 (Sigma-Aldrich, catalog number: P4417 )
Bovine serum albumin fraction V (BSA) (Sigma-Aldrich, catalog number: 10735108001 )
Liquid nitrogen
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
DMSO (Sigma-Aldrich, catalog number: D8418 )
Glycerol, ultra pure (VWR, catalog number: IC800688 )
Sucrose (Sigma-Aldrich, catalog number: S8501 )
Sodium azide (Sigma-Aldrich, catalog number: S2002 )
ProLong gold antifade mountant with DAPI (Thermo Fisher Scientific, catalog number: P36931 )
Lysis buffer (see Recipes)
Equilibration buffer (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)
Dialysis buffer (see Recipes)
Formaldehyde (see Recipes)
Glutamate buffer (see Recipes)
DMEM + HEPES (see Recipes)
PBS + BSA (see Recipes)
Equipment
Water bath
Bacterial Shaker (e.g., Infors HT)
Benchtop Centrifuge (e.g., Beckman Coulter, model: Allegra X-12R )
Centrifuge (e.g., Beckman Coulter, model: Avanti J-26 XP )
Sonicator (e.g., Sonics Vibra Cell)
Tweezer
GSH-Sepharose beads
Magnetic stir bar (Fisher Scientific)
Gel electrophoresis equipment (e.g., Dual minislab, model: ATTO AE6500 )
Fluorescence microscope (e.g., Nikon Eclipse Ti-E model equipped with appropriate filter set for Alexa-Fluor dye)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Munson, M. J. and Ganley, I. G. (2016). Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX). Bio-protocol 6(16): e1903. DOI: 10.21769/BioProtoc.1903.
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Category
Cell Biology > Cell staining > Lipid
Cell Biology > Cell imaging > Fluorescence
Biochemistry > Protein > Labeling
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1,904 | https://bio-protocol.org/exchange/protocoldetail?id=1904&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
MM Michael J. Munson
IG Ian G. Ganley
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1904 Views: 9219
Edited by: Ralph Bottcher
Reviewed by: Neelanjan BoseMarc-Antoine Sani
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity directly in vitro can be a key stage in delineating the action of a particular stimulus.
Keywords: Phosphatidylinositol-3-phosphate VPS34 PIK3C3 Lipid kinase assay TLC
Materials and Reagents
Hamilton syringe (500 μl) (Hamilton, catalog number: 80865 )
Pyrex tubes (12 x 75 mm) (Corning, catalog number: 99445-12 )
Eppendorf flex tubes (Eppendorf, catalog number: 022364111 )
Spin-X columns (0.22 μm) (Corning, Costar®, catalog number: 8161 )
Whatman paper (3 mm) (Sigma-Aldrich, catalog number: 3030-917 )
Silica 60 TLC plates (Merck Millipore Corporation, catalog number: 1.05553.0001 )
TLC spotting capillary tubes, 15.2 cm (VWR, catalog number: 80060-608 )
Polycarbonate membranes, 100 nm pore size (Avanti Polar Lipids, catalog number: 610005 )
Chloroform (VWR International, catalog number: 22707.320 )
Crude liver phosphatidylinositol (PI) (Avanti, catalog number: 840042C )
Methanol (VWR International, catalog number: 20847.307 )
N2 gas tank (99.998% Pure)
VPS34/VPS15 recombinant protein (MRCPPU Reagents, catalog number: DU8692 )
32P-γ-ATP (PerkinElmer, catalog number: NEG002A500UC )
ATP (Sigma-Aldrich, catalog number: A2383 )
Hydrochloric acid (37%) (VWR International, catalog number: 20252.335 )
Glycerol (VWR International, catalog number: 24388.320 )
Sodium dodecyl sulphate (SDS) (VWR International, catalog number: 444464T )
Bromophenol blue (Sigma-Aldrich, catalog number: 114405 )
β-mercaptoethanol
Tris base (VWR International, catalog number: 103157P )
Sodium chloride (VWR International, catalog number: 27810.364 )
Manganese chloride (Sigma-Aldrich, catalog number: M3634 )
CHAPS (Sigma-Aldrich, catalog number: C3023 )
Dithiothreitol (DTT) (ForMedium, catalog number: DTT010 )
Ammonium hydroxide (Sigma-Aldrich, catalog number: 320145 )
ATP mix (5x) (see Recipes)
Kinase assay buffer (see Recipes)
Stop solution (see Recipes)
Sample buffer (1x) (see Recipes)
TLC solvent (see Recipes)
Potassium oxalate (see Recipes)
Equipment
Speedvac (Thermo Fisher Scientific, model: SPD131DDA )
Thermomixer (Eppendorf)
Sonicating Water Bath (Sigma-Aldrich, model: SONOREX Digital 10P )
Mini-extruder set (Avanti, catalog number: 610000 )
Hairdryer
TLC chamber
Fumehood
Phosphoimager (optional) (Fujifilm, model: FLA-2000 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Munson, M. J. and Ganley, I. G. (2016). Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP. Bio-protocol 6(16): e1904. DOI: 10.21769/BioProtoc.1904.
Munson, M. J., Allen, G. F., Toth, R., Campbell, D. G., Lucocq, J. M. and Ganley, I. G. (2015). mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival. EMBO J 34(17): 2272-2290.
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Category
Biochemistry > Protein > Activity
Biochemistry > Lipid > Lipid measurement
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1,905 | https://bio-protocol.org/exchange/protocoldetail?id=1905&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Determination of the H+-ATP Synthase and Hydrolytic Activities
Javier García-Bermúdez*
Cristina Nuevo-Tapioles*
José M. Cuezva
*Contributed equally to this work
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1905 Views: 9041
Edited by: Masahiro Morita
Reviewed by: Daniel KrausAntoine de Morree
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we describe the protocol for the determination of the ATP hydrolytic activity of the enzyme in isolated mitochondria.
Keywords: Mitochondria Oxidative phosphorylation Enzyme activity ATP synthase ATP hydrolase
Part I. The isolation of mitochondria from mouse tissues
Materials and Reagents
10 ml tubes
1.5 ml Eppendorf tubes
Sucrose (Merck Millipore Corporation, catalog number: 107651 )
Ethylenediaminetetraacetic acid disodium salt dehydrate (Sigma-Aldrich, catalog number: ED2SS )
D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
Ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, catalog number: E4378 )
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
Medium A (see Recipes)
Medium H (see Recipes)
Equipment
15 ml Dounce All-Glass tissue grinder (Kimble, catalog number: 885303-0015 )
Beckman Avanti J-25 centrifuge (Beckman Coulter, model: Avanti J-25 )
JA-25.50 rotor, fixed angle, aluminum, single lock lid, 8 x 50 ml, 25,000 rpm, 75,600 x g (Beckman Coulter, catalog number: 363055 )
Bio-VialTM tube, PP, 4 ml, 14 x 55 mm, 0.56 x 2.2 in (Beckman Coulter, catalog number: 566353 )
Centrifuge 5415R (Sigma-Aldrich, Eppendorf®, model: 5415R )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：García-Bermúdez, J., Nuevo-Tapioles, C. and Cuezva, J. M. (2016). Determination of the H+-ATP Synthase and Hydrolytic Activities. Bio-protocol 6(16): e1905. DOI: 10.21769/BioProtoc.1905.
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Category
Biochemistry > Protein > Activity
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1,906 | https://bio-protocol.org/exchange/protocoldetail?id=1906&type=0 | # Bio-Protocol Content
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Peer-reviewed
Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition
Petra Baumgaertner
Daniel E. Speiser
Pedro Romero
NR Nathalie Rufer
Michael Hebeisen
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1906 Views: 18141
Edited by: Masahiro Morita
Reviewed by: Michael EnosEmilie Besnard
Original Research Article:
The authors used this protocol in May 2015
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Abstract
Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).
In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).
Keywords: CD8 T cell Cytotoxic assay Tumor killing assay Chromium-51 Functional avidity
Materials and Reagents
96-well plates, V-shape (Corning, Costar®, catalog number: 3894 )
15 ml Falcon tubes (e.g., Fisher Scientific, catalog number: 14-959-53A )
LumaPlate-96 (PerkinElmer, catalog number: 6006633 )
Bassin or reservoir (e.g., Vitaris, catalog number: 4304-INT or equivalent)
Antigen presenting target cells: e.g., human T2 (CRL-1992), mice P815 (ATCC, catalog number: TIB-64 ) or a characterized tumor cell line [e.g., Me275 (Zippelius et al., 2002)]
Effector cells [(i.e., polyclonal or clonal population of antigen-specific cytotoxic CD8 T cells directly isolated from the blood of patients or healthy donors (Speiser et al., 2008)]
RPMI 1640 (Thermo Fisher Scientific, Gibco®, catalog number: 61870010 )
Fetal bovine serum (FBS) inactivated at 56 °C for 1 h (Thermo Fisher Scientific, Gibco®, catalog number: 10270106 )
Antigenic peptide solution [e.g., NY-ESO-1157-165 peptide (SLLMWITQC)] dissolved in 1x PBS at initial concentration 1 mg/ml
Note: For the controls, an irrelevant peptide solution at 1 mg/ml that is not recognized by the T cells (i.e., any other class I short peptide).
51Chromium solution (Na251CrO4) (2 ml vial containing 2 mCi at a concentration of 1 mCi/ml) (PerkinElmer, catalog number: NEZ030S002MC )
1 M HCl (Sigma-Aldrich, catalog number: 258148 or equivalent )
Equipment
Multichannel pipette, 8 or 12 channels [e.g., Finnpipette F2 10-100 μl, 8 channels (Sigma-Aldrich, catalog number: Z678007 )]
Water bath
Incubator 37 °C, 5% CO2
Standard laboratory desktop centrifuge for 15 ml sample tubes and 96-well plates
Packard TopCount NXT reader (Perkin Elmer, USA)
Software
Prism
Procedure
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How to cite：Baumgaertner, P., Speiser, D. E., Romero, P., Rufer, N. and Hebeisen, M. (2016). Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition. Bio-protocol 6(16): e1906. DOI: 10.21769/BioProtoc.1906.
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Category
Immunology > Immune cell function > Cytotoxicity
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1,907 | https://bio-protocol.org/exchange/protocoldetail?id=1907&type=0 | # Bio-Protocol Content
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Peer-reviewed
Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture
Thuvan Piehler
Nicole Zander
Richard Benjamin
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1907 Views: 6785
Edited by: Soyun Kim
Reviewed by: Pengpeng Li
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
While it is understood that structural damage occurs at the cellular level from the traumatic brain injury event, the effect on functional activity remains largely unknown. Simplified models such as in vitro models of primary explosive blast are critically needed to deconvolute mechanisms of cellular damage. This protocol details an in vitro indoor experimental system setup (Zander et al., 2015) using real military explosive charges to more accurately represent battlefield blast exposure, and probe the effects of primary explosive blast on dissociated neurons.
Keywords: Primary explosive blast model Blast induced traumatic brain injury model Cell culture
Materials and Reagents
12 mm circular glass coverslips (Fisher Scientific, catalog number: 12-545-80 )
24-well plates (Corning, FalconTM, catalog number: 351147 )
Sterile SealPlate® covers (Excel Scientific)
PC12 cells (ATCC, catalog number: CRL-1721 )
Mouse laminin (Corning, catalog number: 354239 )
100% ethanol
Polylysine (Sigma-Aldrich, catalog number: P5899 )
RPMI 1640 (Corning, catalog number: 10-041-CV )
Horse serum (Corning, catalog number: 35-030-CV )
Fetal bovine serum (FBS) (Fisher Scientific, catalog number: SH3007003IH )
Dulbecco Modified Eagle’s Medium (DMEM) (Corning, catalog number: 10-013-CV )
Calf serum (GE Healthcare Life Sciences, HycloneTM, catalog number: SH30072.03 )
Nerve growth factor (NGF) (Corning, catalog number: 356004 )
Spherical 1.77 grams/cm3 cyclotrimethylene trinitramine Class 5 (RDX Class V) charges
HEPES (Fisher Scientific, catalog number: BP299500 )
10-gallon poly (methyl methacrylate) (PMMA)
Calcein-AM (Thermo Fisher Scientific, catalog number: C3099 )
Ethidium homodimer-1 (Thermo Fisher Scientific, catalog number: E1169 )
Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: BP399500 )
Lactate dehydrogenase cytotoxicity assay kit (LDH) (Abnova, catalog number: KA0785 )
RIPA buffer (Alfa Aesar, catalog number: J62189-AP )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
The micro BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23235 )
Hank’s Balanced Salt Solution (HBSS) (Corning, catalog number: 21-022-CV )
Calcein (MP Biomedicals, catalog number: 190167 )
Penicillin/Streptomycin, 50x (Corning, catalog number: 30-001-Cl )
Antibiotic/antimycotic (see Recipes)
Growth/complete medium (see Recipes)
Differentiation medium (see Recipes)
Equipment
Incubator
Aquarium
Three piezoelectric high frequency dynamic pressure sensors (PCB Piezotronics, model: 102A )
Sterile hood
Oven
Zeiss LSM5 Pascal equipped with Epiplan-Neofluar lenses
Confocal laser scanning microscopy (CLSM)
Software
ImageJ v 1.34
Zeiss LSM software (v 4.2.0.121)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Piehler, T., Zander, N. and Benjamin, R. (2016). Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture. Bio-protocol 6(16): e1907. DOI: 10.21769/BioProtoc.1907.
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Category
Neuroscience > Behavioral neuroscience > Cognition
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1,908 | https://bio-protocol.org/exchange/protocoldetail?id=1908&type=0 | # Bio-Protocol Content
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Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry
William M. Oldham
Joseph Loscalzo
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1908 Views: 10703
Edited by: Masahiro Morita
Reviewed by: Shannon RuppertPamela Maher
Original Research Article:
The authors used this protocol in Aug 2015
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Abstract
Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).
Figure 1. Reaction scheme for the derivatization protocol
Keywords: 2-hydroxyglutarate Diacetyl-L-tartaric anhydride Liquid chromatography-mass spectrometry Hydrophilic liquid interaction chromatography
Materials and Reagents
Screw cap 2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 21-403-200 )
D-2-hydroxyglutaric acid disodium salt (Sigma-Aldrich, catalog number: H8378 )
L-2-hydroxyglutaric acid disodium salt (Sigma-Aldrich, catalog number: 90790 )
Sodium lactate (Sigma-Aldrich, catalog number: L7022 )
Water (LC-MS grade) (Thermo Fisher Scientific, catalog number: W6 )
[13C4]-2-oxoglutaric acid disodium salt (Cambridge Isotope Labs, catalog number: CLM-4442 )
Methanol, Optima (LC-MS grade) (Thermo Fisher Scientific, catalog number: A454 )
Diacetyl-L-tartaric anhydride (DATAN) (Sigma-Aldrich, catalog number: 358924 )
Acetonitrile, Optima (LC-MS grade) (Fisher Scientific, catalog number: A955 )
Acetic Acid, Glacial (Fisher Scientific, catalog number: BP2401 )
Formic Acid, Optima (LC-MS grade) (Fisher Scientific, catalog number: A117 )
Ammonium Hydroxide (Fisher Scientific, catalog number: A669 )
Biological samples (see Note 1)
50 mM D2HG and L2HG stock solution (see Recipes)
20 μM 2HG working solution (see Recipes)
10 mM sodium lactate stock solution (see Recipes)
500 μM internal standard (ISTD) stock solution (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)
Equipment
Savant SpeedVac ISS110
Note: This device has been discontinued by Thermo Fisher Scientific.
Isotemp digital dry bath incubator (Fisher Scientific, catalog number: 11-715-125DQ )
Sequant ZIC-HILIC HPLC column (3.5 μm, 100 Å, 2.1 mm internal diameter, 150 mm length) (Merck Millipore Corporation, catalog number: 1504420001 )
Sequant ZIC-HILIC HPLC guard column (Merck Millipore Corporation, catalog number: 1504360001 )
Surveyor autosampler plus
Note: This device has been discontinued by Thermo Fisher Scientific.
Surveyor MS Pump Plus
Note: This device has been discontinued by Thermo Fisher Scientific.
Finnigan LTQ mass spectrometer
Note: This device has been discontinued by Thermo Fisher Scientific.
Software
Xcalibur software (Thermo Fisher Scientific)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Oldham, W. M. and Loscalzo, J. (2016). Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry. Bio-protocol 6(16): e1908. DOI: 10.21769/BioProtoc.1908.
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Category
Biochemistry > Other compound > Acid
Cancer Biology > Cellular energetics > Biochemical assays
Cell Biology > Cell metabolism > Amino acid
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1,909 | https://bio-protocol.org/exchange/protocoldetail?id=1909&type=0 | # Bio-Protocol Content
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Peer-reviewed
Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay
Yubo Tang
Angela Jacobi
Corina Vater
Xuenong Zou
Maik Stiehler
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1909 Views: 9927
Reviewed by: Xiujun Fan
Original Research Article:
The authors used this protocol in Jun 2015
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Abstract
Cell-cell adhesion ensures tight contacts between neighbouring cells, which is necessary for cell segregation, as well as for the morphological and functional differentiation of different tissues. Evidently there are cell-cell recognition systems that make cells of the same type preferentially adherent to one another. Homotypic cell adhesion is particularly important in mediating a range of physiological processes such as cell survival, migration and remodeling of vessels. Thus in the present study we selected two populations of endothelial progenitor cells which are from the same donor to investigate the possible effect of a small molecule compound Icariin on homotypic cell adhesion. Many angiogenic factors can destabilize the organization of intercellular junctions, causing endothelial barrier opening. In the present study, we observed that Icariin treatment reduced the level of VE-cadherin expression in EPCs indicating a decrease in cell-cell adhesion-a proof of the pro-angiogenic effect of Icariin. In summary, the observed loss of homotypic adhesion of EPCs may contribute to the enhanced angiogenic effect exerted by Icariin.
Keywords: Endothelial progenitor cell Intercellular adhesion Cell differentiation
Materials and Reagents
Sterile serological transfer pipettes (Greiner Bio-One, catalog number: 606107 , 607107 , 760107 )
Sterile centrifuge tubes (Greiner Bio-One, catalog number: 188261 )
T75 culture flasks (Corning, catalog number: 353135 )
24-well plates (Corning, Costar®, catalog number: 3524 )
96-well plates (Corning, catalog number: 3596 )
Human bone marrow-derived endothelial progenitor cells (BM-EPCs) [isolated from bone marrow of healthy donors using Ficoll-Paque density gradient (1.077 g/cm3)] (Tang et al., 2015)
Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190169 )
Human fibronectin (BD Biosciences, catalog number: 610077 )
Endothelial cell growth medium MV2 (ECGM2) (PromoCell GmbH, catalog number: 22121 )
Penicillin/streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
Dil-ac-LDL (Thermo Fisher Scientific, catalog number: L3484 )
FITC-UEA-I (Sigma-Aldrich, catalog number: L9006 )
Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
Hoechst 33258 (Santa Cruz Biotechnology, catalog number: sc-394039 )
Calcein AM (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L-3224 )
Ethylene diamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
Trypsin from porcine pancreas (Sigma-Aldrich, catalog number: T4799 )
Endothelial cell growth medium (see Recipes)
0.2 mg/ml Hoechst 33258 (see Recipes)
2 μM calcein-AM (see Recipes)
Equipment
Horizontal centrifuge (Eppendorf, catalog number: 5702RH )
CO2 cell culture incubator (Thermo Fisher Scientific, catalog number: 51026280 )
Flow cytometer (LSR II) (BD Biosciences, catalog number: 342975 )
Scepter 2.0H handheld automated cell counter, w/50 pk of 60 µM Sensors (Merck, Merck Millipore, catalog number: PHCC20060 )
Biological safety cabinets (Thermo Fisher Scientific, catalog number: 51026639 )
Inverted fluorescence microscope (Zeiss, catalog number: Apotome.2 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Tang, Y., Jacobi, A., Vater, C., Zou, X. and Stiehler, M. (2016). Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay. Bio-protocol 6(16): e1909. DOI: 10.21769/BioProtoc.1909.
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Category
Stem Cell > Adult stem cell > Endothelial stem/progenitor cell
Cell Biology > Cell movement > Cell adhesion
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191 | https://bio-protocol.org/exchange/protocoldetail?id=191&type=0 | # Bio-Protocol Content
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c-Fos and Arc Immunohistochemistry on Rat Cerebellum
Soyun Kim
Published: Vol 2, Iss 10, May 20, 2012
DOI: 10.21769/BioProtoc.191 Views: 21409
Original Research Article:
The authors used this protocol in Feb 2011
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Abstract
This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural activity.
Materials and Reagents
c-Fos (raised in mouse, 1:1,000 dilution) (Santa Cruz Biotechnology, catalog number: sc-8074 )
Arc (raised in rabbit, 1:1,000 dilution) (Synaptic Systems, catalog number: 156002 )
c-Fos IHC, Alexa antimouse Fluor 488 (Life Technologies, Invitrogen™, catalog number: 51663A )
Arc IHC, Cy3-conjugated donkey anti-rabbit secondary antibody with 1:500 dilution (Jackson Laboratory, catalog number: 88069 )
Euthasol (Virbac, catalog number: 710101 )
Vectashield (Vector laboratory, catalog number: H-1000 )
PFA powder
0.1 M PBS (pH 7.4)
TritonX-100
NaHPO4
NaH2PO4
Paraformaldehyde (PFA)
NaOH
NaCl
Sucrose
Eythlen glycol (RNAse free) (Sigma-Aldrich, catalog number: E9129 )
Glycerol (RNase free)
Phosphate buffer (PB) (0.2 M stock, pH 7.4) (Solution 1) (see Recipes)
4% Paraformaldehyde (PFA) for transcardial perfusion (Solution 2) (see Recipes)
0.9% Saline (Solution 3) (see Recipes)
30% sucrose in 4% PFA used as cryoprotectant (Solution 4) (see Recipes)
Phosphate buffer saline (PBS) (Solution 5) (see Recipes)
Cryoprotectant (Solution 6) (see Recipes)
Normal donkey serum containing 0.25% Triton-100 (see Recipes)
Primary Antibody (see Recipes)
Secondary Antibody (see Recipes)
Equipment
Fume hood
Water tab
Rib cage
Scissors
Forceps
Clippers
Cannula
Vial
Parafilm
Refrigerator
Tinfoil
Coverslip
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Neuroscience > Behavioral neuroscience > Learning and memory
Developmental Biology > Morphogenesis
Cell Biology > Tissue analysis > Tissue isolation
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1,910 | https://bio-protocol.org/exchange/protocoldetail?id=1910&type=0 | # Bio-Protocol Content
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Peer-reviewed
Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.)
MS Michal Sharabi-Schwager*
MR Mor Rubinstein*
Ms Mazal Ish shalom
RE Ravit Eshed
AR Ada Rozen
AS Amir Sherman
YC Yuval Cohen
Ron Ophir
*Contributed equally to this work
Published: Vol 6, Iss 16, Aug 20, 2016
DOI: 10.21769/BioProtoc.1910 Views: 10141
Edited by: Samik Bhattacharya
Reviewed by: Gazala Ameen
Original Research Article:
The authors used this protocol in Dec 2015
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Abstract
Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high throughput genotyping. We present here a pipeline for developing a reservoir of SNP and SSR markers for Mangifera indica L. as an example for fruit tree crops having no genomic information available. Our pipeline includes de novo assembly of reference transcriptome with MIRA and CAP3 based on reads produced by 454-GS FLX technology; Polymorphic loci discovery by alignment of Illumina resequencing to the transcriptome reference; Identifying a subset of loci that are polymorphic in the entire germplasm collection for downstream diversity analysis by genotyping with Fluidigm technology.
Keywords: SNP discovery Diversity Marker-assisted selection SSR
Background
Considerations of high-throughput sequencing: This pipeline does not include RNA/DNA extraction and other molecular biology lab protocols for next generation sequencing (NGS). It is common to outsourcing NGS. Therefore, it includes DNA preparation for genotyping only. Before describing the pipeline below, we would like to comment about the considerations regarding the sequencing.
Assumption: In this pipeline, we assume a non-model organism which has no genomic infrastructure at all. For marker discovery, one will need a reference and resequencing to discover the polymorphism. The ultimate reference is a genome. However, due to the fact that having a good draft or a complete reference genome is still expensive task our recommendation is to sequence a reference transcriptome from a pool of tissues. The pool of tissues should compensate the unequal gene representation as a result of tissue-specific expression.
Technology: For the purpose of a reference transcriptome sequencing, 454-GS Flx Titanium or any long reads NGS technology is preferred. For marker discovery by resequencing, a pool of genomic DNA (gDNA) from the population under study is a cost-effective solution. Polymorphic loci in such pool are a representative sample of the polymorphic loci in the population. Here the important factor is the reads’ depth which should strive to an average coverage of 50x and no less than 20x. In a case of large genomes the choice of gDNA resequencing might be too expensive to get coverage of 50x. Alternatively, mRNA extraction of a pool of tissues and population individuals would be a cheaper option.
The aim of this protocol is to provide a pipeline (Figure 1) for the bioinformatics and genomics support unit that assist the breeder of a crop which has no genomic information to establish a set of polymorphic SNP and SSR markers. This set can be used for marker-assisted breeding studies as well as for exploring the diversity in the crop’s germplasm collection diversity.
Figure 1. Flowchart of a pipeline for marker discovery. The reference transcriptome here (represented as a database shape) is the link connecting function annotation with genetic variation.
Materials and Reagents
50 ml Falcon tube
Young leaf tissue
Tris (Amresco, catalog number: 77-86-1 )
EDTA (Sigma-Aldrich, catalog number: E5134 )
NaCl (Sigma-Aldrich, catalog number: S3014 )
3% CTAB (Hexadecylrimethylammonium bromide) (Sigma-Aldrich, catalog number: H5882 )
2% polyvinylpyrolidone (PVP) (MW 40,000) (Amresco, catalog number: 9003-39-8 )
1% β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
5 M ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
Chloroform:isoamyl alcohol mix [24:1 (v:v)]
Isopropanol (stored at -20 °C)
Ethanol
RNase A (> 70 Kunit/mg protein, > 20 mg protein/ml) (Sigma-Aldrich, catalog number: R4642 )
Extraction buffer (see Recipes)
TE buffer (see Recipes)
Equipment
65 °C water bath
37 °C water bath/block
IKA-A11 analytical grinding mill (IKA®-Werke GmbH & Co. KG)
Cooled centrifuge (Sorvall RC5plus) with Fixed Angle Rotor (FiberliteTM F13-14 x 50cy) (Thermo Fisher Scientific, catalog number: 096-1450 ).
Agarose gel apparatus
Nanodrop spectrophotometer
Recommended hardware specifications (for bioinformatics pipeline)
CPU
Architecture: x86_64
CPU op-mode(s): 64-bit, 8 cores, Thread(s) per core: 2
Vendor ID: GenuineIntel
CPU MHz: 1596.000
Memory
MemTotal: 48 GB
SwapTotal: 4GB
Software
“Sff_extract” (https://bioinf.comav.upv.es/sff_extract/) – Converting and preprocessing, e.g., adapter removal and base-call clipping 454-GS FLX raw files to text formats (fasta and quality).
Note: Sff_extract is now part of the tool set seq_crumbs (https://bioinf.comav.upv.es/seq_crumbs/)
MIRA (https://sourceforge.net/projects/mira-assembler/) – A multi-pass DNA sequence data assembler/mapper for whole genome and/or transcriptome projects. MIRA is a multi-platforms assembler capable assembling reads from a combination of platforms or from each platform separately.
CAP3 (http://seq.cs.iastate.edu/cap3.html) – CAP3 is for small-scale assembly of sequences with or without quality values.
Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic) – Trimmomatic is a fast, multi-threaded command line tool that can be used to trim and crop Illumina (FASTQ) data as well as to remove adapters.
FASTX (http://hannonlab.cshl.edu/fastx_toolkit/) – Preprocessing, e.g., adapter removal and base-call clipping, short reads (fastq files).
Note: FASTX-Toolkit is a collection of command line tools for Short-Reads FASTA/FASTQ files preprocessing.
Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) – Alignment of short reads to a reference genome/transcriptome.
Samtools (http://www.htslib.org/) – SAM (Sequence Alignment/Map) format is a generic format for storing large nucleotide sequence alignments. SAMTools provide various utilities for manipulating alignments in the SAM format, including sorting, merging, indexing and generating alignments in a per-position format.
Getorf (http://emboss.sourceforge.net/download/) – EMBOSS tool for identification of open reading frame ORF in mRNA sequence.
MIcroSAtellite (MISA) identification tool (http://pgrc.ipk-gatersleben.de/misa) – This tool allows the identification and localization of perfect microsatellites as well as compound microsatellites which are interrupted by a certain number of bases.
SciRoKo (http://kofler.or.at/bioinformatics/SciRoKo/index.html) – A tool for fast whole-genome microsatellite search. For example, the whole rice genome may be searched in 55 sec.
VarScan (http://varscan.sourceforge.net/) – VarScan is a platform-independent mutation caller for targeted, exome, and whole-genome resequencing data generated on Illumina, SOLiD, Life/PGM, Roche/454, and similar instruments.
Data analysis
Data
454-GS FLX Titanium mRNA contigs were deposited in transcriptome shotgun assembly (TSA) repository of NCBI: accession No. GBJO00000000 (Sherman et al., 2015).
Illumina short reads were deposited in short reads archive (SRA) of NCBI: experiment accession No. SRX651793 GBJO00000000 (Sherman et al., 2015).
De novo transcriptome assembly
Raw sequence reads of the 454-GS FLX Titanium platform were pre-processed by “Sff_extract” (https://bioinf.comav.upv.es/sff_extract/) and arguments for removing the adaptors and clipping the poly-A were applied.
De novo assembly with MIRA 3.2 (Chevreux et al., 2004)
Reduction of contig variability (merging transcript variants) by running Cap3 and creating super-contigs
Note: Cap3 is downloaded separately from MIRA (see Software list section).
Note: mango.fasta and mango.qual are output files of MIRA, created in the mango_d_results directory.
Filtering out contigs with length less than 200 bp
Refer to the fasta file from here on as reference.transcriptome.contigs.fasta
Functional annotation
Identifying the coding region to annotate marker position, i.e., inside or outside coding sequence. Finding open reading frames (ORFs) by the “getorf” program of the EMBOSS package (Rice et al., 2000). The longest ORF with start and stop codons was chosen for each contig (-find 1) with a minimum cutoff of 50 amino acids (-minisize 150).
Note: The argument (-minimize) is given in base pairs (50 bp x 3 = 150 bp).
Reference transcriptome contigs annotation to connect variability with functionality using Blast 2GO (Gotz et al., 2008).
Blast2GO GUI options:
Start → load sequences (e.g., fasta)
Blast → Run Blast Description Annotator
Mapping → Run mapping
Annot → Run annotation
InterPro → Run interproscan
SNP and SSR discovery
Adapter removal and low-quality base pairs clipping are performed by Trimmomatic (Bolger et al., 2014) and FASTX (http://hannonlab.cshl.edu/fastx_toolkit/).
Note: Optional but highly recommended if the alignment is performed on RNA-Seq.
Use R1 and R2 of trimmed pair files, i.e., sample_name_pair_L001_R1.fastq, sample_name_pair_L001_R2.fastq for downstream analysis.
Alignment of resequencing of Illumina HiSeq-2000 reads to the transcriptome reference with bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).
Running samtools (http://www.htslib.org/) and VarScan (Koboldt et al., 2009) for SNP discovery
Note: The criteria for selecting an SNP subset are dependent on the project. However, a few criteria are advisable to ensure confident SNP loci in any project:
No other SNPs in the flanking regions (100 bp each side) to enable of primer design for further analyses (Absolute differences of the SNP value between the previous and next values in ‘Position’ column should > 100).
Only one SNP per reference-transcriptome contig (unique value at ‘Chrom’ column).
Bi-allelic confidence (‘SamplesHet’ value > 0).
SSR discovery within the contigs the reference transcriptome.
MIcroSAtellite (MISA) identification tool (http://pgrc.ipk-gatersleben.de/misa) and SciRoKo (Kofler et al., 2007) are run with default parameters.
Find the intersection between two tables by importing them into MS-Access or SQLite and run a SQL inner join command on contig-name, motif, and start-position.
Genotyping with Fluidigm
Large-scale genomic DNA extraction for sample genotyping isolated from young leaves
Young developing Mango (Mangifera indica L.) leaves were collected from the orchard, frozen in liquid nitrogen and stored at -80 °C until used.
β-mercaptoethanol was added to extraction buffer which was pre-heated to 65 °C in a pre-warmed water bath.
2 g of young leaf tissue was ground to a fine powder using IKA-A11 analytical grinding mill or with a mortar and pestle.
Ground tissue was transferred to a 50 ml Falcon tube and extracted with 15 ml pre-warmed extraction buffer. Extraction was performed by incubation for 30 min at 65 °C, with occasional mixing of the tube.
15 ml of chloroform:isoamyl alcohol mix (24:1, v:v) was added to tubes. Samples were mixed and centrifuged at 17,000 x g for 10 min at 4 °C.
The aqueous phase was transferred to a new 50 ml tube, and re-extracted with 15 ml of chloroform:isoamyl alcohol mix (24:1, v:v). Centrifugation was performed as above.
The aqueous phase was transferred to a new tube. 1 volume of ice-cold isopropanol was added, tubes were mixed and DNA was precipitated by centrifugation at 17,000 x g for 20 min at 4 °C.
Supernatant was carefully disposed of. Pellet was washed with 70% ice-cold ethanol, and centrifuged at 17,000 x g for 10 min at 4 °C.
Supernatant was carefully disposed of. Pellet was left to dry at room temperature until it turns translucent), and suspended in 3 ml of TE buffer.
DNA solution was treated with 3 μl RNase A, and incubated for 30 min in 37 °C.
DNA is precipitated by adding 1/10 volume of 5 M ammonium acetate, and 2/5 volumes of cold 100% ethanol. Tubes are mixed and centrifuged at 17,000 x g for 10 min at 4 °C.
The supernatant was carefully disposed of. Pellet was washed with 70% ice-cold ethanol, and centrifuged at 17,000 x g for 10 min at 4 °C.
Pellet is air dried and final DNA is suspended in 200 μl of double distilled water or TE buffer.
DNA concentration and quality is analyzed on a 0.7% TAE agarose gel and with a Nanodrop spectrophotometer.
Genotyping on Fluidigm – EP1 Fluidigm standard protocols for FR96.96 chip with four no-template controls (NTCs) instead of one.
Briefly, the protocol is divided into two major sub-protocols – pre-amplification and the assay itself:
First, specific target amplification (STA) protocol is performed to have an approximately equal proportion from each target by running the following steps:
1) Preparing the 10x SNPtype STA. Primer pool for 96 assays.
2) Performing STA on a PCR machine.
3) Dilution of samples (the outcome will be used in stage 4 of the second part).
Second, the assay of genotyping by specific target primers is performed in a Fluidigm 96.96 dynamic genotyping array on the EP1 platform as follow:
1) Priming the 96.96 Dynamic ArrayTM IFC.
2) Preparing SNPtype assays mixes.
3) Preparing 10x Assays.
4) Preparing Sample Pre-Mix and Samples.
5) Loading the Chip.
6) Using the FC1TM Cycler.
7) Using the EP1TM Reader Data Collection Software.
8) Extracting the data for downstream bioinformatics analysis.
The full protocol description can be found at (http://www.mscience.com.au/upload/pages/fluidigmtech/fluidigm-snp-genotyping-user-guide-151112.pdf).
Filtering qualified SNPs for diversity analysis
Fluidigm genotype calls are divided into four categories by visual inspection:
Filtering out SNPs with a Category ≥ 2 (Table 1).
Filtering out SNPs with more than 10% no calls.
Filtering out samples with more than 33% no calls.
Filtering out markers with PIC < 0.1.
Filtering out markers with more than 90% of the samples have the same call, i.e., segregating exactly the same.
Filtering out markers with less than 2 samples in each genotype.
Leaving only one marker from each pair of linked markers (R^2 > 0.7).
Leaving only one sample from group of sample having identical genotype. (Identity ≥ 0.95)
*Notes:
These steps can be performed with any programming language, e.g., R, python, perl, C, etc. or SQL.
PIC is calculated as PIC = 1- ∑ pi^2; i = a, A
R^2 is calculated as r^2 = D^2/(p1*p2*q1*q2); D = (p11*p22)-(p12p21 p11,p22,p12,p21 are the proportions of all possible combinations of two bi-allelic loci.
Table 1. Quality scores of each locus genotyping calls given by visual inspection.
Recipes
Extraction buffer
100 Tris, pH 8
20 M EDTA
1.5 M NaCl
3% hexadecylrimethylammonium bromide (CTAB)
2% polyvinylpyrolidone (PVP)
1% β-mercaptoethanol
All solution except β-mercaptoethanol are dissolved by stirring over several hours, and autoclaved. β-mercaptoethanol is added just prior to tissue extraction.
TE buffer
10 mM Tris, pH 8.0
1 mM EDTA
Acknowledgments
The protocol has been developed in a study which was supported by the Chief Scientist of Ministry of Agriculture and Rural Development [Grant No.: 203-0859-12].
References
Bolger, A. M., Lohse, M. and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114-2120.
Bowtie - An ultrafast memory-efficient short read aligner. JOHNS HOPKINS University.
Chevreux, B., Pfisterer, T., Drescher, B., Driesel, A. J., Muller, W. E., Wetter, T. and Suhai, S. (2004). Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res 14(6): 1147-1159.
Fluidigm SNP Genotyping Guide. Fluidigm.
FASTX-Toolkit. Hannonlab.
Gotz, S., Garcia-Gomez, J. M., Terol, J., Williams, T. D., Nagaraj, S. H., Nueda, M. J., Robles, M., Talon, M., Dopazo, J. and Conesa, A. (2008). High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36(10): 3420-3435.
Koboldt, D. C., Chen, K., Wylie, T., Larson, D. E., McLellan, M. D., Mardis, E. R., Weinstock, G. M., Wilson, R. K. and Ding, L. (2009). VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics 25(17): 2283-2285.
Kofler, R., Schlotterer, C. and Lelley, T. (2007). SciRoKo: a new tool for whole genome microsatellite search and investigation. Bioinformatics 23(13): 1683-1685.
MIcroSAtellite identification tool.
Reading/writing/editing/indexing/viewing SAM/BAM/CRAM format. Samtools.
Rice, P., Longden, I. and Bleasby, A. (2000). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16(6): 276-277.
Sff_extract. Bioinformatics at COMAV.
Sherman, A., Rubinstein, M., Eshed, R., Benita, M., Ish-Shalom, M., Sharabi-Schwager, M., Rozen, A., Saada, D., Cohen, Y. and Ophir, R. (2015). Mango (Mangifera indica L.) germplasm diversity based on single nucleotide polymorphisms derived from the transcriptome. BMC Plant Biol 15: 277.
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Category
Plant Science > Plant molecular biology > DNA
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > DNA extraction
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1,911 | https://bio-protocol.org/exchange/protocoldetail?id=1911&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Isolation of Joint-infiltrating Cells
Aoi Akitsu
Yoichiro Iwakura
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1911 Views: 10473
Edited by: Ivan Zanoni
Reviewed by: Achille BroggiMarco Di Gioia
Original Research Article:
The authors used this protocol in May 2014
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The authors used this protocol in:
May 2014
Abstract
Infiltration of leukocytes into joints is one of the main features of autoimmune inflammatory arthritis. Here, we describe the protocol for isolation of joint-infiltrating cells in mice. This protocol is useful to analyze cell surface antigens and intracellular cytokines by flow cytometry.
Keywords: Joint Arthritis Inflammation Autoimmune disease
Materials and Reagents
27-gauge needle (TERUMO, catalog number: NN-2719S )
1 ml syringe (TERUMO, catalog number: SS-01T )
6-well plate (Coring, Falcon®, catalog number: 353046 )
50 ml tube
70 μm cell strainer (Coring, Falcon®, catalog number: 352350 )
10 ml syringe (TERUMO, catalog number: SS-10SZ )
Mice (adult > 6 weeks, any sex, any strain)
Hyaluronidase (60 mg/ml stock) (Sigma-Aldrich, catalog number: H3506 )
Collagenase type VIII (10 mg/ml stock) (Sigma-Aldrich, catalog number: C2139 )
RPMI 1640 (Sigma-Aldrich, catalog number: R8758 )
Fetal bovine serum, heat inactivated (Biowest, catalog number: S1820-500 )
HANK’s solution “Nissui”② (NISSUI PHARMACEUTICAL, catalog number: 05906 )
NaHCO3 (NAKARAI TESQUE, catalog number: 31212-25 )
NaN3 (NAKARAI TESQUE, catalog number: 31208-82 )
Digestion medium (see Recipes)
FACS solution (see Recipes)
Equipment
Surgical scissors and tweezers
Mini-shaker 3D (Biosan, catalog number: BS-010151-AAK )
Refrigerated centrifuge
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Akitsu, A. and Iwakura, Y. (2016). Isolation of Joint-infiltrating Cells. Bio-protocol 6(17): e1911. DOI: 10.21769/BioProtoc.1911.
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Category
Immunology > Immune cell isolation > Leukocyte
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1,912 | https://bio-protocol.org/exchange/protocoldetail?id=1912&type=0 | # Bio-Protocol Content
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Peer-reviewed
BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry
BQ Bo Qiu
MS M. Celeste Simon
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1912 Views: 60097
Edited by: Lee-Hwa Tai
Reviewed by: Yong TengVanesa Olivares-Illana
Original Research Article:
The authors used this protocol in Jun 2015
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The authors used this protocol in:
Jun 2015
Abstract
Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis (Greenberg et al., 2011). For example, neutral lipid storage is enhanced upon increased synthesis or uptake of lipid species. On the other hand, extracellular signals can enhance the release of lipid species packaged within neutral LDs. Thus, the investigation of topics involving lipid metabolism may require the assessment of cellular neutral lipid content. In this protocol, we describe the use of the fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) to facilitate quantification of neutral lipid content by flow cytometry and observation of LDs by microscopy.
Keywords: Neutral lipid Lipid droplet BODIPY
Materials and Reagents
35 mm cell culture dish/well
Flow cytometry tube with cell strainer cap (Corning, catalog number: 352235 )
15 ml conical tube
35 μm filter
FACS tube
Glass slides
Circular cover slip (12 mm #1 circular) (VWR International, catalog number: 101415-528 )
Cell line of interest
Note: For this protocol, we utilize the A498 clear cell renal cell carcinoma cell line (ATCC, catalog number: A-498 ), but this method can be readily performed with other cell lines.
Oleic acid (OA) (100 mg/ml with 10% BSA in DPBS) (Sigma-Aldrich, catalog number: O3008 )
Bovine serum albumin (BSA) (10% in DPBS, low endotoxin, fatty acid free, suitable for cell culture, sterile-filtered) (Sigma-Aldrich, catalog number: A1595-50ML )
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D3922 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco®, catalog number: 10010031 )
Trypsin-EDTA (0.25%) with phenol red (Thermo Fisher Scientific, Gibco®, catalog number: 25200-056 )
HEPES
NaCl
CaCl2
Dulbecco’s modified Eagle’s medium with high glucose and L-glutamine (Thermo Fisher Scientific, Fisher Scientific, catalog number: SH3024301 ) or other medium appropriate for cell line of interest
Fetal bovine serum (FBS) (Gemini Bio-Products, FoundationTM, catalog number: 900-108 ) or other FBS suitable for cell line of interest
Note: For this protocol, cells were grown in DMEM + 10% FBS.
4% paraformaldehyde (PFA)
Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36935 )
Collagen I, rat tail (100 mg) (Corning, catalog number: 354236 )
2 μM BODIPY staining solution (see Recipes)
10x flow cytometry buffer (see Recipes)
Note: 10x buffer can also be purchased [10x Annexin binding buffer (BD, catalog number: 556454 )].
Equipment
Forceps
Flow cytometer equipped with 488 mm laser and filter sets for measuring FITC or GFP (i.e., 533/30)
Note: In this study, a BD Accuri C6 instrument was used.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Qiu, B. and Simon, M. C. (2016). BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry. Bio-protocol 6(17): e1912. DOI: 10.21769/BioProtoc.1912.
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Category
Cell Biology > Cell staining > Lipid
Biochemistry > Lipid > Lipid measurement
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1,913 | https://bio-protocol.org/exchange/protocoldetail?id=1913&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
This protocol has been corrected. See the correction notice.
Peer-reviewed
Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton
Tianzi Chen
BZ Baolong Zhang
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1913 Views: 27854
Edited by: Tie Liu
Reviewed by: Hideyuki TakahashiZhanwu Dai
Original Research Article:
The authors used this protocol in May 2015
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Original research article
The authors used this protocol in:
May 2015
Abstract
Drought stress negatively affects cotton plant growth and induces various biochemical and physiological responses in cotton plants. Proline content and antioxidant enzymes are thought to be associated with maintaining the structure of cellular components or with protecting cellular function. Study of cotton plant responses towards drought stress and investigation of the mechanism of drought tolerance are helpful to develop drought tolerant cotton plants. Here, we describe a protocol to investigate cotton plant response towards drought stress through measurements of biochemical parameters including antioxidant enzyme activities, proline content and malondialdehyde (MDA) content.
Materials and Reagents
Centrifuge tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-150-C-S )
Gossypium barbadense seeds
Soil mix (vermiculite:humus = 1:1) (http://www.xing-nong.cn/ProductList.Asp?SortID=207)
1/2 MS media salt without sugar and agar (Hopebio, catalog number: HB8469-12 )
Sodium phosphate dibasic anhydrous (Na2HPO4) (Sinopharm Chemical Reagent, catalog number: 20040618 )
Sodium phosphate monobasic anhydrous (NaH2PO4) (Sinopharm Chemical Reagent, catalog number: 20040818 )
ddH2O
Liquid nitrogen
Ice
Ninhydrin (C9H4O3·H2O) (Sinopharm Chemical Reagent, catalog number: 30130212 )
Trichloroacetic acid (TCA) (Sinopharm Chemical Reagent, catalog number: 80132618 )
5-sulphosalicylic acid dihydrate (Sinopharm Chemical Reagent, catalog number: 10021516 )
L-proline (Genview, catalog number: FL259 )
Thiobarbituric acid (TBA) (Sigma-Aldrich, catalog number: T5500 )
Sodium hydroxide (NaOH) (Sinopharm Chemical Reagent, catalog number: 10019762 )
EDTA-2Na (Sigma-Aldrich, catalog number: 27285 )
L-methionine (Sigma-Aldrich, catalog number: V900487 )
Nitroblue tetrazolium (NBT) (Beijing Dingguo Changsheng Biotechnology, catalog number: JN213 )
Riboflavin (Sigma-Aldrich, catalog number: 47861 )
Guaiacol (Sigma-Aldrich, catalog number: G5502 )
30% hydrogen peroxide (H2O2) (Sinopharm Chemical Reagent, catalog number: 10011208 )
Ethanol (Sinopharm Chemical Reagent, catalog number: 10009259 )
Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251 )
1-chloro-2, 4-dinitrobenzene (CDNB) (Sigma-Aldrich, catalog number: C38702 )
Acetic acid (Sinopharm Chemical Reagent, catalog number: 10000208 )
1/2 MS solution (see Recipes)
Na2HPO4 stock solution (see Recipes)
NaH2PO4 stock solution (see Recipes)
PBS (pH 7.0) (see Recipes)
2.5% acid-ninhydrin (see Recipes)
3% sulphosalicylic acid (see Recipes)
10% TCA (see Recipes)
0.25% TBA (see Recipes)
PBS (pH 7.8) (see Recipes)
1 mM EDTA-2Na (see Recipes)
130 mM methionine (see Recipes)
750 μM NBT (see Recipes)
20 μM riboflavin (see Recipes)
0.2% guaiacol (see Recipes)
PBS (pH 6.5) (see Recipes)
5 mM GSH (see Recipes)
1.5 mM CDNB (see Recipes)
Equipment
Plant growth chamber (25 °C, 16 h/8 h photoperiod, 10,000 lux, 50-60% humidity)
Mortar and pestle
Plastic pots (8 cm diameter and 12 cm depth) (http://www.lyyyb.com/yingyangbo.html)
Electronic balance (Sartorius, model: BAS124S-CW )
ddH2O purification system (Xiamen RSJ Scientific Instruments, model: Spring-S10 )
Ice making machine (Xueke, model: IMS-40 )
Centrifuge (Eppendorf, model: 5810 R )
Spectrophotometer (Analytik Jena, model: ScanDrop® 250)
Water bath (Changzhou Aohua Instrument, model: HH-1 )
Pipette, 2-20 µl (Eppendorf, Eppendorf Research® plus, model: 3120000038 )
Pipette, 20-200 µl (Eppendorf, Eppendorf Research® plus, model: 3120000054 )
Pipette, 100-1,000 µl (Eppendorf, Eppendorf Research® plus, model: 3120000062 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, T. and Zhang, B. (2016). Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton. Bio-protocol 6(17): e1913. DOI: 10.21769/BioProtoc.1913.
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Category
Plant Science > Plant physiology > Abiotic stress
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Freezing tissue for a period of time affects extraction
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is it poissable measure by plate reader rather by spectrophotometer ScanDrop 250
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1,914 | https://bio-protocol.org/exchange/protocoldetail?id=1914&type=0 | # Bio-Protocol Content
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Peer-reviewed
Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint
CV Céline Vallot
CR Claire Rougeulle
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1914 Views: 8682
Original Research Article:
The authors used this protocol in May 2015
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May 2015
Abstract
We developed a FISH-based method to directly assess chromosome-wide transcriptional activity, thereby enabling the visualization of the actively transcribed fraction of a chromosome at the single-cell level. We applied this method to probe the activity of X-chromosomes and its instability in the context of human embryonic stem cells and cancer cells.
Keywords: Transcription Immuno-fluorescence Chromosome-wide
Materials and Reagents
24-well plates (Sigma-Aldrich, catalog number: Z707791-126EA )
13 mm round coverslips (Thermo Fisher Scientific, catalog number: 174950 )
Glass slides
Filter (0.2 μm)
Filtration unit (Merck Millipore, catalog number: SCGPS05RE )
Human embryonic stem cells (H9, WIBR2 and HUES1) and cancer cells (TCCSUP and RT112)
Matrigel
PBS solution (Life Technologies)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
PIPES (Sigma-Aldrich, catalog number: P6757 )
NaOH
Triton X-100 (Sigma-Aldrich, catalog number: X100 )
Ribonucleoside vanadyl complex (New England Biolabs, catalog number: S1402 )
Paraformaldehyde (PFA) (16%, EM grade) (VWR International, catalog number: 100503-916 )
EtOH
Human Cot-1 DNA (Life Technologies)
Sodium acetate anhydrous (NaOAc) (Sigma-Aldrich, catalog number: W302406 )
Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
Rubber cement
20x SSC (Sigma-Aldrich, catalog number: 93017 )
20 mg/ml BSA (New England Biolabs, catalog number: B9000S )
Dextran sulfate (Sigma-Aldrich, catalog number: 67578 )
Formamide (Sigma-Aldrich, catalog number: 47670 )
Mounting medium (Vector Laboratories, catalog number: H-1200 )
Cy3-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-OR )
FITC-labelled human X-Chromosome paint (Metasystem, catalog number: D-0323-050-FI )
CSK buffer (see Recipes)
3% PFA/PBS solution (see Recipes)
Hybridisation buffer (2x) (see Recipes)
Washing solution (50% formamide/2x SSC) (see Recipes)
Denaturating solution (70% formamide/2x SSC) (see Recipes)
Equipment
Hybridisation table (Boekel Scientific, model: 240000 )
Shake 'N BakeTM hybridisation oven (Boekel Scientific, model: 136400 )
Centrifuge (VWR, Ependorf®, model: 5417R )
Millivac-mini vacuum pump (Merck Millipore, catalog number: XF5423050 )
Ependorf® Thermomixer® R (Sigma-Aldrich, catalog number: T3317 )
Note: This product has been discontinued.
Fluorescent microscope (motorized stage) (Leica, model: DMI-6000 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Vallot, C. and Rougeulle, C. (2016). Single-cell Visualization of Chromosome Transcriptional Territories by RNA-paint. Bio-protocol 6(17): e1914. DOI: 10.21769/BioProtoc.1914.
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Category
Stem Cell > Embryonic stem cell > Cell-based analysis
Molecular Biology > DNA > Gene expression
Molecular Biology > RNA > Transcription
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1,915 | https://bio-protocol.org/exchange/protocoldetail?id=1915&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Cell Wall Stress Assay for Fusarium oxysporum
Elena Pérez-Nadales
AP Antonio Di Pietro
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1915 Views: 9631
Edited by: Zhaohui Liu
Reviewed by: Emmanuel Zavalza
Original Research Article:
The authors used this protocol in Aug 2015
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Aug 2015
Abstract
In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).
Materials and Reagents
Round (90 mm diameter) or square (120 x 120 mm) Petri dishes
Autoclavable, filtration material [e.g., Miracloth (Merck Millipore, catalog number: 475855 )]
Calcofluor white fluorescent brightener (Sigma-Aldrich, catalog number: F-3543 )
Congo red (Sigma-Aldrich, catalog number: 860956 )
Distilled water
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Potatoes
Cloth strainer
Potato dextrose agar (PDA) (Scharlab, catalog number: 01-483-500 )
Yeast extract (Merck Millipore, catalog number: 1119261000 )
Peptone (Merck Millipore, catalog number: 1119311000 )
Glucose (VWR, Normapur®, catalog number: 50-99-7 )
Agar (BD, BactoTM, catalog number: 214010 )
MgSO4·7H2O (Merck Millipore, catalog number: 1058865000 )
KH2PO4 (Merck Millipore, catalog number: 1048731000 )
KCl (Merck Millipore, catalog number: 1049330500 )
NaNO3 (Merck Millipore, catalog number: 1065371000 )
Sucrose (Merck Millipore, catalog number: 1076875000 )
Agar (Thermo Fisher Scientific, OxoidTM, catalog number: LP0011 )
2-(N-morpholino) ethanesulfonic acid (MES), monohydrate (Sigma-Aldrich, catalog number: 69892 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
Potato dextrose broth (PDB) (see Recipes)
CFW and CR stock solutions (see Recipes)
CFW stock solutions (20 mg/ml)
CR stock solutions (50 mg/ml)
Fungal growth agar medium (see Recipes)
YPD agar medium
PDA agar medium
Puhalla’s minimal agar medium (MM)
Note: CFW and CR are hazardous and potentially carcinogenic so caution must be taken to avoid skin contact or inhalation of these compounds.
Equipment
Protection gloves and coat
Flasks and magnetic stirrer for preparation of solutions
Neubauer chamber for cell counting
Microwave
Autoclave
Fungal growth incubator
Water bath
Laminar flow hood
Heat plate and cooking pot to boil potatoes
pH meter
Note: No other special equipment is required.
Software
SPSS 15.0 for Windows® (LEAD Technologies Inc.)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Pérez-Nadales, E. and Di Pietro, A. (2016). In vitro Cell Wall Stress Assay for Fusarium oxysporum. Bio-protocol 6(17): e1915. DOI: 10.21769/BioProtoc.1915.
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Category
Microbiology > Antimicrobial assay > Antifungal assay
Microbiology > Microbe-host interactions > Fungus
Biochemistry > Other compound > Antimicrobial
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1,916 | https://bio-protocol.org/exchange/protocoldetail?id=1916&type=0 | # Bio-Protocol Content
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Peer-reviewed
Simple Methods for Screening and Statistical Analysis of Leaf Epidermal Cells in Dicotyledonous Plants
JT Jian-Jun Tao
SC Shou-Yi Chen
JZ Jin-Song Zhang
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1916 Views: 12483
Edited by: Marisa Rosa
Reviewed by: Joëlle Schlapfer
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
Leaf epidermal cell size and number are positively correlated to leaf area. Stomata are specialized epidermal cells vital for gas exchange and water transpiration. So, observation and statistical analysis of the leaf epidermal cells are valuable for the study of leaf development and response to environmental stimulus. The classical method is using the scanning electron microscope (SEM), which is an expensive and time-consuming method, thus makes the large-scale screening of epidermis impractical. Here we provide simple but effective methods (agarose-based epidermal imprinting and tape-based epidermis tearing) for solving this problem without using the SEM.
Keywords: Epidermal cell Stomata Epidermal imprinting Epidermis tearing ImageJ
Materials and Reagents
Glass slide (Leica, model: 3809299 )
Scotch tape (1/2") (3M, Scotch®, model: 600 )
Tobacco (one month old) and soybean seedlings (two weeks old)
Distilled water
Low melting point agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16520-050 )
3% agarose solution (see Recipes)
Equipment
Thermostatic water bath (40 °C)
Pointed tweezer (Tweezerman, model: 4091833 )
Refrigerator (4 °C) (Haier, model: HT18TS77SP )
Optical microscope (Olympus, model: BX51 )
Objective used on microscope (Olympus, model: UPlanFI )
Software
ImageJ (version 1.38x or above)
Procedure
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Category
Plant Science > Plant cell biology > Cell structure
Plant Science > Plant physiology > Tissue analysis
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Nail polish method is not appropriate for stomata imprint of wild tomato because of its dense trichome. Can you suggest me any other easy method?
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1,917 | https://bio-protocol.org/exchange/protocoldetail?id=1917&type=0 | # Bio-Protocol Content
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Peer-reviewed
Protein Expression, Purification and Crystallization of the Sxl-Unr-msl2 Ribonucleoprotein Complex
Janosch Hennig
MS Michael Sattler
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1917 Views: 9080
Edited by: Arsalan Daudi
Reviewed by: Longping Victor Tse
Original Research Article:
The authors used this protocol in Nov 2014
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The authors used this protocol in:
Nov 2014
Abstract
This protocol describes the expression, purification and crystallization of a ternary protein-protein-RNA complex, consisting of the two RNA recognition motifs (RRMs) of Sex-lethal (Sxl), the first of five cold shock domains of Upstream-of-N-Ras (Unr), and an 18-nucleotide region of msl2 mRNA, called the F fragment (Hennig et al., 2014).The biological role of the complex is the translational repression of msl2 mRNA, preventing the formation of the dosage compensation complex and subsequent 2-fold hypertranscription of X-linked genes in Drosophila females. As orthologous RRM-containing proteins and Unr exist in humans, similar complexes potentially also form during translational repression in vertebrates. The protocol describes the in vitro assembly of the complex and its purification followed by crystallization for X-ray crystallography structure determination. Part of the protocol has been published elsewhere (Hennig et al., 2013 and 2014), but some parts are described here in more detail.
Materials and Reagents
Membrane dialysis tubing Spectra/Por 6 prewetted, 1.1 ml/cm 3,500 Da cut-off (Spectrum, Spectra/Por®, catalog number: 132590 )
Membrane dialysis tubing Spectra/Por 6 prewetted, 1.1 ml/cm 10,000 Da cut-off (Spectrum, Spectra/Por®, catalog number: 128118 )
Amicon® Ultra-15 centrifugal filter unit with Ultracel-3 membrane (Merck Millpore, catalog number: UFC900308 )
Amicon® Ultra-15 centrifugal filter unit with Ultracel-10 membrane (Merck Millpore, catalog number: UFC901008 )
Amicon® Ultra-4 centrifugal filter unit with Ultracel-3 membrane (Merck Millpore, catalog number: UFC800324 )
Amicon® Ultra-4 centrifugal filter unit with Ultracel-10 membrane (Merck Millpore, catalog number: UFC801024 )
EasyXtal 15-well tool (Qiagen, catalog number: 132006 )
0.22 µm filter
96-well plate
SS34 centrifugation tubes
Escherichia coli (E. coli) Bl21 (DE3)from stocks (stored at -80 °C)
pTRX-Sxl and pTRX-CSD plasmids (available upon request)
Tobacco etch virus (TEV) protease (1 mg/ml stock solution, in-house production)
Precision plus protein all blue prestained protein standard (Bio-Rad Laboratories, catalog number: 1610373 )
LB medium
Kanamycin (33 mg/ml stock solution, stored at -20 °C)
Isopropyl-β-D-1-thiogalactopyranoside (IPTG) (1 M stock solution)
Ni-NTA resin (Qiagen, catalog number: 30230 )
Sodium phosphate (molecular biology grade)
Sodium chloride (molecular biology grade)
Potassium phosphate (molecular biology grade)
Imidazole (molecular biology grade, > 99%)
β-mercaptoethanol
Sodium azide
Ammonium sulfate (molecular biology grade, > 99% purity)
18-mer RNA based on E fragment of msl2 mRNA: UUU UUU UGA GCA CGU GAA (synthesized) (IBA) (UniProt Consortium, catalog number: P50534 )
Lithium sulfate (X-ray crystallography grade, > 99% purity)
PEG 3350 (Sigma-Aldrich, catalog number: 39713 )
Dithiothreitol (DTT) (> 99% purity)
Glycerol (Sigma-Aldrich, catalog number: 49767 )
Lysis buffer (see Recipes)
Wash buffer A (see Recipes)
Wash buffer B (see Recipes)
Elution buffer (see Recipes)
NMR buffer (see Recipes)
Running buffer (see Recipes)
Equipment
Erlenmeyer flasks (500 ml)
FPLC protein purifier
Superdex S75 gel filtration size-exclusion chromatography column HiLoad 26/600 (GE Healthcare)
Nanodrop system
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hennig, J. and Sattler, M. (2016). Protein Expression, Purification and Crystallization of the Sxl-Unr-msl2 Ribonucleoprotein Complex. Bio-protocol 6(17): e1917. DOI: 10.21769/BioProtoc.1917.
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Category
Biochemistry > Protein > Structure
Biochemistry > RNA > RNA-protein interaction
Biochemistry > Protein > Isolation and purification
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1,918 | https://bio-protocol.org/exchange/protocoldetail?id=1918&type=0 | # Bio-Protocol Content
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Peer-reviewed
Salinity and Drought Treatment Assays in Kenaf (Hibiscus cannabinus L.)
Xiaoping Niu
MC Meixia Chen
AT Aifen Tao
Jianmin Qi
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1918 Views: 9651
Edited by: Arsalan Daudi
Reviewed by: Jian ChenMohan TC
Original Research Article:
The authors used this protocol in Dec 2015
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The authors used this protocol in:
Dec 2015
Abstract
Salinity and drought are the two main factors that cause fiber yield and quality losses in kenaf. It is reported that salinity and drought can affect more than 10% of arable land and cause a global decline in the average yields of major crops by more than 50%. Therefore, understanding plant tolerance of drought and salinity is of fundamental importance and has become the focus of intensive research. This protocol describes a simple and reproducible protocol to imitate natural salinity and drought stress under soil conditions. Even though the water-culture method is most frequently used for salinity and drought treatments, the soil-culture method in this study was more applicable to studying natural stress conditions.
Keywords: Salinity Drought Kenaf (Hibiscus cannabinus L.)
Materials and Reagents
Sterile Petri dishes (JET, catalog number: TCD010090 )
Sterile filter paper (Sigma-Aldrich, catalog number: Z241091 )
Seeds: kenaf (cv Fuhong 992) [conserved in the Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops of Fujian Agriculture and Forestry University (China)].
Sterile water (sterilized for 20 min at 121 °C/1 atm using an autoclave)
Sterile soil (Fafard® growing mix 1-PV/C-1PV, Sun Gro Horticulture Canada Ltd.) (sterilized for 30 min at 121 °C/1 atm using an autoclave)
Sodium hypochlorite (NaClO) solution (15%) (Sinopharm Chemical Reagent, catalog number: 7681-52-9 )
Potassium nitrate (KNO3) (Sinopharm Chemical Reagent, catalog number: 7757-79-1 )
Ammonium dihydric phosphate (NH4H2PO4) (Sinopharm Chemical Reagent, catalog number: 7722-76-1 )
Ammonium nitrate (NH4NO3) (Sinopharm Chemical Reagent, catalog number: 6484-52-2 )
Calcium nitrate [Ca(NO3)2·4H2O] (Sinopharm Chemical Reagent, catalog number: 13477-34-4 )
Magnesium sulphate (MgSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10034-99-8 )
Ethylene diamine-N,N bis (2 hydroxyphenylacetic acid) Ferric sodium complex (Fe-EDDHA) (Sinopharm Chemical Reagent, catalog number: 16455-61-1 )
Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10043-35-3 )
Manganese chloride (MnCl2·4H2O) (Sinopharm Chemical Reagent, catalog number: 7773-01-5 )
Zinc sulphate (ZnSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 7446-20-0 )
Copper sulphate (CuSO4·5H2O) (Sinopharm Chemical Reagent, catalog number: 7758-98-7 )
Sodium molybdate (Na2MoO4·2H2O) (Sinopharm Chemical Reagent, catalog number: 10102-40-6 )
Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 7647-14-5 )
PEG6000 (Sigma-Aldrich, catalog number: 81260 )
5% (v/v) sodium hypochlorite(see Recipes)
Hoagland solution (see Recipes)
200 mM sodium chloride (NaCl) solution (see Recipes)
20% (w/v) PEG6000 solution (see Recipes)
Equipment
Plastic pots (10 cm diameter and 8 cm depth)
Clean bench (Suzhou AirTech , AirTech®, model: SW-CJ-1FD )
Growth chamber (Yihengyiqi Shanghai, Blue pard®, model: MGC-300A )
Autoclave (TOMY, model: SX-500 )
Precision balance (± 0.001)
Timer control
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Niu, X., Chen, M., Tao, A. and Qi, J. (2016). Salinity and Drought Treatment Assays in Kenaf (Hibiscus cannabinus L.). Bio-protocol 6(17): e1918. DOI: 10.21769/BioProtoc.1918.
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Category
Plant Science > Plant physiology > Abiotic stress
Plant Science > Plant physiology > Plant growth
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1,919 | https://bio-protocol.org/exchange/protocoldetail?id=1919&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Cycloheximide Assays to Measure Protein Degradation in vivo in Plants
Jonathan Gilkerson
RT Raymond Tam
AZ Aimee Zhang
KD Kate Dreher
JC Judy Callis
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1919 Views: 18581
Edited by: Tie Liu
Reviewed by: Yuko Kurita
Original Research Article:
The authors used this protocol in Jun 2015
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The authors used this protocol in:
Jun 2015
Abstract
The half-life of a protein is a characteristic property, and can be modulated by post-translational modifications, changes in subcellular localization, and/or interaction with other proteins or ligands. As one determinant of its steady-state level, a protein’s degradation represents an important distinguishing attribute relevant to its biological function. Because protein longevity cannot be elucidated from bioinformatics analyses, it must be determined empirically. Here we describe two approaches for in vivo half-life determination in plants: 1. pooled-seedling degradation assays monitoring either tagged versions of the protein (luciferase fusions or other epitope tags) or following the endogenous protein; 2. single-seedling degradation assays using luciferase fusion proteins. The advantages of these approaches are their simplicity and low cost.
Keywords: Protein degradation Cycloheximide Proteolysis Half-life Degradation assays
Materials and Reagents
60 x 15 mm sterile Petri dishes (Corning, Falcon®, catalog number: 353002 )
Microfuge tubes
Pipet tips
White polystyrene flat-bottom 96-well plates (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-566-619 )
PVDF (polyvinylidene fluoride) Western protein blotting membrane (Sigma-Aldrich, Amersham HybondTM, catalog number: GE10600023 )
Clear adhesive plate film (USA-Scientific, catalog number: 2921-7810 )
X-ray film (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34091 )
Plant lines expressing protein of interest, either from transgene or from endogenous locus
Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C1988 )
Primary antibody [example, anti-HA-HRP (3F10) (Sigma-Aldrich, Roche, catalog number: 12158167001 )]
Secondary antibody (horseradish peroxidase-linked) (Bio-Rad Laboratories, catalog number: 1662408EDU )
Tween-20 (Sigma-Aldrich, catalog number: P9416 )
Bleach (sodium hypochlorite) (VWR International, Chlorox®, catalog number: 89501-620 ) or from a grocery or drugstore, such as Chlorox bleach
Murashige and Skoog basal salts with micronutrients (Sigma-Aldrich, catalog number: M5524 )
Sucrose
MES [2-(N-morpholino)ethanesulfonic acid] free acid (Merck Millipore, Calbiochem®, catalog number: 475893 )
Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
Thiamine-HCl (Sigma-Aldrich, catalog number: T4625 )
Pyroxidine-HCl (Sigma-Aldrich, catalog number: P9755 )
Myo-inositol (Sigma-Aldrich, catalog number: I1525 )
Potassium hydroxide (KOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P250 )
Bacto agar (BD, Difco, catalog number: 214010 )
Parafilm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-374-10 )
Liquid nitrogen
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
Ethylenediaminetetraacetic acid (EDTA), disodium dehydrate (Thermo Fisher Scientific, catalog number: S311 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Tricine (Sigma-Aldrich, catalog number: T0377 )
Magnesium chloride hexahydrate (MgCl2) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M33 )
BSA (bovine serum albumin) for protein standard curve (Sigma-Aldrich, catalog number: A7030 )
Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A2383 )
Tris base (Sigma-Aldrich, catalog number: T1503 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
IGEPAL CA-630 [Octylphenoxy poly (ethyleneoxy) ethanol] (Sigma-Aldrich, catalog number: I8896 ), a nonionic, non-denaturing detergent replacement for Nonidet P-40
MG132 (Benzyloxycarbonyl-L-Leucyl-L-Leucyl-L-Leucinal) (Peptides International, catalog number: IZL-3175-v )
Complete mini protease inhibitor EDTA-free tablet (Sigma-Aldrich, Roche, catalog number: 4693159001 )
Protein concentration assay (Bio-Rad Laboratories, catalog number: 5000006 )
Chemiluminescent western blot development kit (SuperSignal West Pico or Dura Extended duration substrate) (Thermo Fisher Scientific, catalog number: 34077 and 34075 , respectively)
D-Luciferin potassium salt (Gold Biotechnology, catalog number: LUCK-100 )
Growth media (GM) (see Recipes)
Luciferase extraction buffer (see Recipes)
Luciferase assay buffer (see Recipes)
Protein extraction buffer for Western blot assays (see Recipes)
Equipment
Plate reading spectrophotometer for protein determination assay (Thermo Fisher Scientific)
Refrigerated centrifuge (Eppendorf)
SDS-PAGE gel apparatus and power supply (Thermo Fisher Scientific)
Porcelain mortar and pestle for protein extraction (Thermo Fisher Scientific)
Western transfer apparatus (Thermo Fisher Scientific)
Plate reading luminometer (Berthold Technologies)
Flatbed scanner (Hewlett-Packard)
X-ray film developer, either a set of tanks or trays, or an automatic machine
Orbital shaker for Western blot incubation (Thermo Fisher Scientific)
Software
Excel and another statistical package such as GraphPad Prism or Stata
NIH ImageJ 1.36 (http://rsb.info.nih.gov/ij/)
ImageQuant 1.0 software (http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_16016/29000605)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Gilkerson, J., Tam, R., Zhang, A., Dreher, K. and Callis, J. (2016). Cycloheximide Assays to Measure Protein Degradation in vivo in Plants. Bio-protocol 6(17): e1919. DOI: 10.21769/BioProtoc.1919.
Gilkerson, J., Kelley, D. R., Tam, R., Estelle, M. and Callis, J. (2015). Lysine residues are not required for proteasome-mediated proteolysis of the auxin/indole acidic acid protein IAA1. Plant Physiology 168(2): 708-720.
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Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Degradation
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192 | https://bio-protocol.org/exchange/protocoldetail?id=192&type=0 | # Bio-Protocol Content
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Immunofluorescence Staining on Mouse Embryonic Brain Sections
Xuecai Ge
Published: Vol 2, Iss 11, Jun 5, 2012
DOI: 10.21769/BioProtoc.192 Views: 24448
Original Research Article:
The authors used this protocol in Jan 2010
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Abstract
This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections.
Keywords: Immunofluorescence staining Brain section Paraformaldehyde Antibody Cryostat
Materials and Reagents
Paraformaldehyde (PFA)
It can be ordered as 16% PFA from Electron Microscopy Sciences (catalog number: 15710 ), and diluted to 4% before use. However, we usually make the 4% solution from the powder (Sigma-Aldrich, catalog number: 158127 ). Here is how to make 4% PFA:
In a 500 flask, weigh 4 g PFA in 80 ml PBS.
Stir and heat the mixture on a magnetic heater. Keep the temperature below 60 °C during the entire process to avoid breakdown of the polymers.
When temperature is close to 60 °C, add a few drops of 10 N sodium hydroxide. The mixture will gradually become clear.
Filter the solution into a new flask and cool down to room temperature.
Neutralize with hydrochloric acid to pH 7.
Add PBS to 100 ml.
O.C.T. (TFM Tissue Freezing Medium, TFM-5, clear color)
Phosphate buffered saline (PBS)
Goat serum
Triton X-100
Secondary antibodies:
Cy2 (or cy3, or cy5)-conjugated goat-anti-mouse (or rabbit) IgG.
(Jackson ImmunoResearch, F (ab) 2 fragments of affinity-purified antibody)
Hoechst33258 (Sigma-Aldrich, catalog number: 861405 )
Antifade reagent (Life Technologies, Invitrogen™, catalog number: P36930 )
Ethanol
4 N hydrochloric acid
Sodium hydroxide
Blocking buffer (see Recipes)
Equipment
Plastic cryomolds (Ted Pella, catalog number: 27110 )
Cryostat sectioning machine
SuperFrost Plus slides (VWR International, catalog number: 48311-703 )
Dark humidified chamber: Made from a square petri dish containing kimwipes that are soaked in water. Wrap the chamber with aluminum foil to protect the slides from light.
PAP pen
500 flask
Ice bucket
Magnetic heater
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Neuroscience > Development > Immunofluorescence
Cell Biology > Tissue analysis > Tissue isolation
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1,920 | https://bio-protocol.org/exchange/protocoldetail?id=1920&type=0 | # Bio-Protocol Content
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Peer-reviewed
Identification of RNA-binding Proteins
KM Kazuya Masuda
TK Tadamitsu Kishimoto
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1920 Views: 13339
Edited by: Antoine de Morree
Reviewed by: Zhen ShiKate Hannan
Original Research Article:
The authors used this protocol in Jun 2013
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The authors used this protocol in:
Jun 2013
Abstract
This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).
Figure 1. Schematic diagram of procedure (A-H). Flow chart of experimental procedure is indicated at A-H.
Figure 2. Linearization of plasmids by restricted enzyme. The plasmid is cut at restriction sites adjacent to its cloning element.
Keywords: RNA-protein interactions 5-bromo-UTP-incorporated RNA Mass spectrometry Pull-down assay
Materials and Reagents
Microcentrifuge tubes (1.5 to 2.0 ml)
Desalting MobiSpin columns (MoBiTec, catalog number: M105035F )
DNA template (e.g., pBluescript vector encoding the target sequences such as non-coding elements IL-6 3’UTR and TNF-α 3’UTR)
Reagents for in vitro transcription kits (TAKARA BIO, catalog number: 6140 )
5-bromouridine 5’-triphosphate (sodium salt) (Cayman Chemical, catalog number: 18140 )
50 mM ATP solution
50 mM GTP solution
50 mM CTP solution
50 mM UTP solution
RNaseOUTTM recombinant ribonuclease inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019 )
T7 RNA polymerase
DNase I (RNase free) (New England Biolabs, catalog number: M0303S )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: MSSAFE )
Protease inhibitor cocktail (NACALAI TESQUE, catalog number: 0 4080 )
TRIzol® Reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596-026 )
Anti-BrdU antibody (IIB5) (Abcam, catalog number: ab8152 )
Protein G Sepharose 4 fast flow (GE Healthcare, catalog number: 17061801 )
Nuclease-free PBS (NACALAI TESQUE, catalog number: 14249 )
Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: 4387936 )
Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
Chloroform (NACALAI TESQUE, catalog number: 08401-65 )
DEPC water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9916 )
96% ethanol or 70% ethanol (NACALAI TESQUE, catalog number: 09666-85 )
NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, catalog number: 78833 )
0.25% trypsin-EDTA solution (Thermo Fisher Scientific, catalog number: 25200056 )
1 M Tris-HCl, pH 7.4 (NACALAI TESQUE, Gibco®, catalog number: 35436-01 )
5 M NaCl (NACALAI TESQUE, catalog number: 31320-05 )
0.1 M spermidine (Sigma-Aldrich, catalog number: S2626-1G )
10% Nonidet(R) P-40 (NACALAI TESQUE, catalog number: 25223-04 )
0.5 M EDTA (NACALAI TESQUE, catalog number: 06894-14 )
1 M MgCl2 (NACALAI TESQUE, catalog number: 20942-34 )
1% Tween 20 (NACALAI TESQUE, catalog number: 35624-15 )
PBS, pH 7.0 (NACALAI TESQUE, catalog number: 14249-24 )
1% SDS (NACALAI TESQUE, catalog number: 30562-04 )
Coomassie brilliant blue (CBB) (Thermo Fisher Scientific, catalog number: 20278 )
10x transcription buffer (see Recipes)
Bead washing buffer (see Recipes)
RNA-binding buffer (see Recipes)
RNA-protein wash buffer (see Recipes)
Lysis buffer (see Recipes)
Elution buffer (see Recipes)
Equipment
MALDI-QIT-TOF (Shimadzu Europa GmbH, model: AXIMA Resonance )
Microcentrifuge capable of reaching up to 16,000 x g
Vortex mixer
Centrifuge capable of reaching up to 2,000 x g
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Masuda, K. and Kishimoto, T. (2016). Identification of RNA-binding Proteins. Bio-protocol 6(17): e1920. DOI: 10.21769/BioProtoc.1920.
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Category
Biochemistry > RNA > RNA-protein interaction
Biochemistry > Protein > Isolation and purification
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1,921 | https://bio-protocol.org/exchange/protocoldetail?id=1921&type=0 | # Bio-Protocol Content
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Peer-reviewed
Olfactory Bulb (OB) Transplants
Maria Savvaki
Domna Karagogeos
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1921 Views: 7427
Original Research Article:
The authors used this protocol in Dec 2015
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The authors used this protocol in:
Dec 2015
Abstract
Transplantation in mouse brain slices is a powerful tool in order to study axon targeting and migrational events during development. Taking advantage of donors and recipients belonging to different genotypes, this technique allows researchers to assess the contribution of donor and/or recipient tissue by performing various combinations and to study cell-autonomous functions or effects that are influenced by the recipient’s environment (Bastakis, et al., 2015). Here we describe the transplantation procedure on sagittal brain slices containing olfactory bulb (OB). Specifically, we have transplanted the proximal-to-the-cortex part of dorsal OB to the same region on a recipient slice. Transplanted slices can be cultured for up to 3 days before their morphology is disfigured due to growth in 3D. Re-sectioning of these slices allows for a more detailed immunohistochemical analysis.
Materials and Reagents
Millicell cell culture insert, 30 mm, hydrophilic PTFE, 0.4 µm (Merck Millipore, catalog number: PICM0RG50 )
Disposable transfer pipettes (SARSTEDT, catalog number: 86.1172.001 )
5 ml stripette disposable serological pipette (Sigma-Aldrich, catalog number: CLS4051 )
50 ml Falcon tube
Sterile 24-well plate
Stainless surgical blade (Swann Morton, Lance, model: No. 24 )
Vibratome blade (Campden Instruments, catalog number: 752-1-SS )
60 mm culture dishes
Straight triangle insert pin (Fine Science Tools, catalog number: 26007-04 )
Stainless steel minutien pins 0.10 mm (Fine Science Tools, catalog number: 26002-10 )
Filters (0.2 μm, 0.45 μm)
Mouse embryos at embryonic days E13.5-E15.5
Leibovitz’s L-15 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11415-064 )
SeaPlaqueTM low melting temperature agarose (Lonza, catalog number: 50101 )
Sterile water
100 units/ml penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
NaCl (Merck Millipore, catalog number: 106404 )
KCl (Merck Millipore, catalog number: 104936 )
Na2HPO4 (Merck Millipore, catalog number: 106586 )
NaH2PO4 (Sigma-Aldrich, catalog number: S-0751 )
NaOH (Merck Millipore, catalog number: 106498 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
DMEM/F12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21041-025 )
Fetal bovine serum (FBS) (Merck Millipore, Biochrom, catalog number: S0115 )
1x glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-038 )
N-2 supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12348-017 )
B-27 supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Leibovitz’s L-15/penicillin-streptomycin medium (see Recipes)
3% SeaPlaque agarose in Leibovitz’s L-15 (see Recipes)
10x phosphate buffer solution (PBS) (see Recipes)
10 M NaOH (see Recipes)
Sterile 1x PBS (0.1 M PBS) with 100 units/ml penicillin-streptomycin (see Recipes)
25% glucose in 1x PBS (see Recipes)
DMEM/F12 plus medium (see Recipes)
Neurobasal/ B-27 plus medium (See Recipes)
4% paraformaldehyde (PFA) (see Recipes)
Equipment
37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, model: 3110 )
Dissecting microscope (Leica Microsystems, model: MZ125 )
2 Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10 )
Moria perforated spoon (Fine Science Tools, catalog number: 10370-17 )
250 ml glass beaker
Two-in-One micro spatula (Fine Science Tools, catalog number: 10091-12 )
Leica vibratome (VT 1000S)
Nickel plated pin holder (Fine Science Tools, catalog number: 26018-17 )
Note: All surgical tools should be UV-sterilized before use.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Savvaki, M. and Karagogeos, D. (2016). Olfactory Bulb (OB) Transplants. Bio-protocol 6(17): e1921. DOI: 10.21769/BioProtoc.1921.
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Category
Developmental Biology > Cell growth and fate > Neuron
Cell Biology > Tissue analysis > Tissue isolation
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1,922 | https://bio-protocol.org/exchange/protocoldetail?id=1922&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues
SJ Supak Jenkitkasemwong
Chia-Yu Wang
Mitchell D. Knutson
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1922 Views: 8296
Edited by: Masahiro Morita
Reviewed by: G. Bhanuprakash ReddyYong Teng
Original Research Article:
The authors used this protocol in Jul 2015
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The authors used this protocol in:
Jul 2015
Abstract
Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.
Keywords: NTBI TBI 59Fe Tissue iron uptake Iron overload
Materials and Reagents
1 ml TB syringe with 27-gauge needle (BD Biosciences, catalog number: 309623 )
Plastic wrap
3.5” x 3.5” weigh boat (Thermo Fisher Scientific, Fisher Scientific, catalog number: 8732113 )
1.5-ml microcentrifuge tubes
0.45 µm filter unit (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-740-65B )
AmiconTM Ultra-15 centrifugal filter units (Merck Millipore, catalog number: UFC903024 )
5 ml tube
Adult mice (> 6 weeks, any strain) (e.g., Balb/cJ)
59FeCl3 (Perkin Elmer, catalog number: NEZ037500UC )
Note: The minimum specific activity of 59Fe we have used to obtain decent signal from our gamma counter was 0.2 Ci/mmol at the concentration of ~3 µCi/µl, which gave whole body counts per minute of ~100,000 per each animal. Our gamma counting efficiency is about 10% for 59Fe.
Nitrilotriacetic acid (NTA) (Sigma-Aldrich, catalog number: N9877-100G )
HEPES (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC172570250 )
Tris (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233-500 )
Sodium citrate dihydrate (Thermo Fisher Scientific, Fisher Scientific, catalog number: S279-500 )
Ferric chloride hexahydrate (FeCl3·6H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I88-100 )
Hydrochloric acid (HCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A144-212 )
Ethanol
Human apo-transferrin (Sigma-Aldrich, catalog number: T2252 )
PBS (VWR, Corning®, catalog number: 21-040-CM )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S2713 )
FisherbrandTM absorbent underpads (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-206-64 )
Radioactive decontaminating solution (Thermo Fisher Scientific, Fisher Scientific, catalog number: NC9633347 )
20 mM Fe-NTA (1:4) (see Recipes)
Ferric citrate solution (see Recipes)
59Fe-labeled ferric citrate solution (see Recipes)
59Fe-labeled transferrin (see Recipes)
0.9% NaCl solution (see Recipes)
Equipment
Gamma counter (PerkinElmer, catalog number: 2480-0010 )
The program for 59Fe detection was set using the following parameters:
Counting window: Dynamic-keV
Peak position: 1292 keV
Low boundary: 1020 keV
High boundary: 1400 keV
Threshold: 20%
Max. assay deviation: 20%
Max. normalization deviation: 50%
Warning assay deviation: 15%
Significant CPM/keV: 10
Counting spectrum type: many peaks
Gamma counting tubes (5 x 28 mm) and RIA racks (PerkinElmer, catalog number: 1480-151 )
Prior to gamma counting, empty gamma counting tubes are measured to ensure no 59Fe contamination (i.e., < 25 CPM = background).
Mouse restrainer (Braintree Scientific, catalog number: MTI STD )
Basic surgical kit for small animals
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Jenkitkasemwong, S., Wang, C. and Knutson, M. D. (2016). Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues. Bio-protocol 6(17): e1922. DOI: 10.21769/BioProtoc.1922.
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Category
Biochemistry > Other compound > Ion
Cell Biology > Cell metabolism > Other compound
Cancer Biology > Cellular energetics > Biochemical assays
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1,923 | https://bio-protocol.org/exchange/protocoldetail?id=1923&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Method to Analyze Local and Systemic Effects of Environmental Stimuli on Root Development in Plants
Neil E. Robbins II
José Dinneny
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1923 Views: 9896
Edited by: Renate Weizbauer
Reviewed by: Alizée MalnoeHonghong Wu
Original Research Article:
The authors used this protocol in Jun 2014
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Jun 2014
Abstract
Root development in vascular plants is innately tied to the environment. However, relatively little attention has been paid toward understanding the spatial scales at which the root perceives and responds to external stimuli. While some environmental signals elicit global responses that affect root system architecture, others may have more localized effects. We have observed that various developmental processes can be induced or suppressed along the circumference of the main root depending on local contact with available water in a process termed hydropatterning (Bao et al., 2014). Our studies of hydropatterning indicate that the root can detect and respond to certain external stimuli at the resolution of the diameter of a single organ. In order to characterize developmental patterning at this spatial scale, we developed a procedure to vary environmental inputs across the circumferential axis of the root in vitro using agar media. Roots are grown between two blocks of agar media in a “sandwich”. Local environmental conditions can be varied depending on the composition of the media on either side. Stimuli that act locally can be distinguished from those that act systemically based on the developmental response of the root. Here we describe the overall method and provide an example of how it can be used to analyze lateral root patterning in Zea mays (maize) in response to an external water potential gradient. We also discuss how the method can be used more broadly for other plant species and environmental treatments.
Keywords: Plant-environment interactions Root development Lateral roots Agar sandwich Water stress
Materials and Reagents
Germination paper (76 lb heavy weight seed germination paper) (Anchor Paper Company)
120 x 120 x 17 mm square plastic Petri plates (USA Scientific, catalog number: 5668-8102 )
Aluminum foil
Tri-fold paper towels (von Drehle Corporation, catalog number: 548-W )
Silicone rubber (food-grade, high-temperature, 1/32” thickness, 50A medium hardness) (McMaster-Carr, catalog number: 86045K131 )
Razor blades
50 ml conical tubes
Filter paper, Grade 40 (Sigma-Aldrich, Whatman®, catalog number: Z241261 )
Parafilm (VWR International, Bemis, catalog number: PM999 )
Distilled water
MS salts (Caisson Laboratories, catalog number: MSP01-50LT )
MES hydrate (Sigma-Aldrich, catalog number: M2933 )
Potassium hydroxide (1 M aqueous solution)
Agar (BD, DifcoTM, catalog number: 214530 )
Mannitol (PhytoTechnology Laboratories, catalog number: M562 )
Maize kernels (B73 inbred line)
Bleach (8.25% sodium hypochlorite solution)
Tween 20 (Sigma-Aldrich, catalog number: P2287 )
70% (v/v) ethanol in water
Superglue (Krazy Glue, catalog number: KG582 )
Control agar media (see Recipes)
Treatment agar media (see Recipes)
Maize kernel sterilization solution (see Recipes)
Equipment
Glass bottles (500 ml capacity or greater)
pH meter
Autoclave
Laminar flow hood
4 °C cold room or refrigerator
Glass beakers (500 ml capacity or greater)
Hot water bath or hot plate
Thermometer
Timer
Forceps
Plant growth chamber (Percival, model number: CU41L4 )
Permanent marker
Microwave
Ruler
Computer equipped to run R or comparable statistical analysis software
Software
R or comparable statistical analysis software (download link: https://cloud.r-project.org/)
Procedure
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How to cite：Robbins II, N. E. and Dinneny, J. (2016). A Method to Analyze Local and Systemic Effects of Environmental Stimuli on Root Development in Plants. Bio-protocol 6(17): e1923. DOI: 10.21769/BioProtoc.1923.
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Category
Plant Science > Plant physiology > Abiotic stress
Plant Science > Plant physiology > Plant growth
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1,924 | https://bio-protocol.org/exchange/protocoldetail?id=1924&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts
Rodolphe Soret
Nicolas Pilon
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1924 Views: 7483
Edited by: Jia Li
Reviewed by: Guillermo GomezJyotiska Chaudhuri
Original Research Article:
The authors used this protocol in Dec 2015
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Dec 2015
Abstract
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These “enteric” neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret et al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.
Keywords: Enteric nervous system Mouse Neural crest cells Cell migration ex vivo
Materials and Reagents
Petri dishes (Corning, catalog number: 70165-102 )
24-well plate
Nitrocellulose filter (Merck Millipore, catalog number: GSWP01300 )
8-chamber slides (ibiTreat μ-slide) (ibidi GmbH, catalog number: 80826 )
Mature mice (≥ 2-month old)
Isoflurane for inhaled anesthesia (Henry Schein Animal Health, catalog number: 050031 )
70% ethanol
1x phosphate-buffered saline (PBS)
DMEM/Ham’s F-12 (WISENT, catalog number: 319-085-CL )
Fetal Bovine Serum (WISENT, catalog number: 920-040 )
Penicillin/streptomycin (WISENT, catalog number: 450-201-EL )
Equipment
Dumont #5 dissection forceps (Fine Science Tools, catalog number: 11251-20 )
Dumont #7 dissection forceps (Fine Science Tools, catalog number: 11274-20 )
Dissection scissors (Moria Spring Scissors) (Fine Science Tools, catalog number: 15396-01 )
Dissecting stereomicroscope (Leica Microsystems, model: M125 )
CO2 cell culture incubator (Sanyo Scientific, model: MCO-18AIC )
Note: This product has been discontinued.
Infinity-2 camera (Lumenera Corporation) mounted on a fluorescent stereomicroscope (Leica Microsystems, model: M205FA )
Software
ImageJ software
Procedure
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How to cite：Soret, R. and Pilon, N. (2016). Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts. Bio-protocol 6(17): e1924. DOI: 10.21769/BioProtoc.1924.
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Category
Neuroscience > Nervous system disorders > Animal model
Developmental Biology > Cell growth and fate > Neuron
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1,925 | https://bio-protocol.org/exchange/protocoldetail?id=1925&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Object-context Recognition Memory Test for Mice
SK Sofia Kanatsou
HK Harm Krugers
Published: Vol 6, Iss 17, Sep 5, 2016
DOI: 10.21769/BioProtoc.1925 Views: 9652
Edited by: Soyun Kim
Reviewed by: Edgar Soria-GomezXi Feng
Original Research Article:
The authors used this protocol in Aug 2015
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Abstract
The object in-context (OIC) task is a variant of the widely used object recognition (OR) task (Dix and Aggleton, 1999). The OIC task makes use of the fact that rodents have a natural tendency to explore novel environments and objects. The hippocampus appears to play a major role in the OIC task (much more so than in the original OR task), where animals should be able to distinguish between two familiar objects of which one is in a different context from the training trial (Ennaceur and Aggleton,1997; Bermudez-Rattoni et al., 2005; Albasser et al., 2009; Roozendaal et al., 2010; Banks et al., 2014; Bermudez-Rattoni, 2014). Recognition memory encompasses a number of additional components, such as an item's associations with its context, place, etc. (Bussey et al., 1999, 2000). Here, we describe a version of the OIC task in mice, based on earlier reports (Dix and Aggleton, 1999; Eacott and Norman, 2004; Balderas et al., 2008; Barsegyan et al., 2014; Kanatsou et al., 2015a; Kanatsou et al., 2015b).
Keywords: Learning Memory Hippocampus Mice Cognition
Materials and Reagents
The protocol was approved by the committee on Animal Health and Care from the University of Amsterdam, the Netherlands (permit number: DED 291).
Standard sawdust bedding (just enough to cover the bottom of the box)
Note: The amount of bedding should be enough to cover the bottom of the context to reduce anxiety behavior. However, this may interfere with other ethological behaviours (e.g., digging), which in turn makes the mouse not to focus on the object but instead with the bedding. You can also try without bedding, but you need to test this before yourself.
Mouse, C57BL/6J strain, male and female, 2-4 months old, group housed (2-4 mice per group)
Note: Mice were kept in a temperature and humidity controlled facility (21.5-22 °C, with humidity between 40 and 60%) on a 12 h light/dark cycle (lights on at 8:00 a.m.) with food and water available ad libitum.
70% ethanol (v/v)
Note: Clean the objects using 70% ethanol. Air dry for 1 min.
Equipment
Apparatus
For context we used four blue-colored plastic boxes of identical size (W x L x H = 33 x 54 x 37 cm) with or without visual cues on the walls. As visual cues we used tape in white color drawing cues in the walls (Figure 1).
Note: Consider a large open field apparatus for this test, because the displacement of an object is more noticeable compared to a small size apparatus, while at the same time you provide the mouse with the ability to explore the extra cues in the box.
Figure 1. Images for Objects and contexts. A. Day 2: training in context A (Lego objects) for 10 min. B. Day 2: training in context B (bottle objects) - > 10 min. C. Day 3: testing in context B (replaced bottle with Lego from context A) - > 10 min.
Objects
We used blocks of Lego and/or small bottles (Figure 1).
Note: The objects should be approximately of equal size to the mouse or slightly bigger. The two set of objects should be different enough to be distinguished by the mice. The objects are recommended to be made of hard plastic or glass, so they are resistant to biting. Both two set of objects should be of approximately equal size.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kanatsou, S. and Krugers, H. (2016). Object-context Recognition Memory Test for Mice. Bio-protocol 6(17): e1925. DOI: 10.21769/BioProtoc.1925.
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Category
Neuroscience > Behavioral neuroscience > Cognition
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1,926 | https://bio-protocol.org/exchange/protocoldetail?id=1926&type=0 | # Bio-Protocol Content
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Peer-reviewed
Paper Roll Culture and Assessment of Maize Root Parameters
AA Adel H. Abdel-Ghani*
Darlene L. Sanchez*
BK Bharath Kumar
TL Thomas Lubberstedt
*Contributed equally to this work
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1926 Views: 12077
Edited by: Tie Liu
Reviewed by: Sriema L. WalawageArsalan Daudi
Original Research Article:
The authors used this protocol in May 2015
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Abstract
Selection for genotypes with a vigorous root system could enhance the adaptation of maize under water and nutrient deficit soils. Although extensive genetic variation for root architecture has been reported (Kumar et al., 2012; Abdel-Ghani et al., 2014; Kumar et al., 2014; Pace et al., 2015), root traits have been seldom considered as selection criteria to improve yield in maize, mainly because characterization of root morphology in the field is laborious, inaccurate and time consuming (Tuberosa and Salvi, 2007). Characterization of root traits under hydroponic conditions in this case has the advantage of screening a high number of genotypes in a small space (in a growth chamber) within a short period of time (2-3 weeks). Thus, it saves the time and effort required for screening maize genotypes with vigorous root systems and might be helpful to monitor root development at different growth stages.
Materials and Reagents
Regular weight (brown) germination paper 48.5” x 36.5”, custom-sized to 12” x 24” (Anchor Paper Company, catalog number: SD3836S )
Small kitchen wire mesh strainer/sieve with handle (20 cm diameter)
Small plastic cups, measuring boats (FisherbrandTM Hexagonal Polystyrene Weighing dishes) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 02-202-101 ), or double-faced filter paper for drying seed- should be the same number as the number of entries
Waterproof pencil art grip aquarelle black (Faber-Castell, catalog number: 114299 ) and permanent marker (Sharpie®, Fine Point Permanent Marker, black)
Note: Black marker works better as other colors fade faster.
Plastic tags (5” x 5/8”) (International Greenhouse, catalog number: CN-1000 ) for labeling (optional if rolls are labeled)
Rubber bands (OfficeMax Extra Long Rubber bands, or any other brand)
Glassine bags (Seedburo S411 shoot bags, treated, 4” x 2-1/2” x 11”)
Personal protective items: latex gloves, lab coat, closed shoes, mask goggles
Maize seeds: Genotypes used in this protocol are pure lines obtained from the North Central Regional Plant Introduction Station in Ames, Iowa (Abdel-Ghani et al., 2013).
Notes:
Seeds should be multiplied under the same conditions to avoid differences due to the environment on the seed size.
Seeds should display high germination percentages to keep a similar number of biological replications within experimental units.
Chlorox® solution (6% sodium hypochlorite), household bleach (USA)
Deionized sterile distilled water (ddH2O)
Potassium nitrate (KNO3) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P-263-500 )
Calcium nitrate [Ca(NO3)2] (Thermo Fisher Scientific, Fisher Scientific, catalog number: C109-3 )
Monopotassium phosphate or potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP-362-500 )
Magnesium sulfate (Thermo Fisher Scientific, Fisher Scientific, catalog number: M65-500 )
Iron from iron chelate [Fe-EDTA, (Sigma-Aldrich, catalog number: E6760-100G ), Fe-DTPA, or Fe-EDDHA]
Monocalcium phosphate or calcium phosphate monobasic [Ca(H2PO4)2] (MP Biomedicals, catalog number: 193803 )
Calcium sulfate dihydrate (CaSO4·2H2O) (MP Biomedicals, catalog number: 191414 )
Potassium sulfate (K2SO4) (Sigma-Aldrich, catalog number: P0772-1kg )
Boric acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP168-1 )
Manganese chloride-4 hydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 221279-100g )
Zinc sulfate-7 hydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
Copper sulfate-5 hydrate (Thermo Fisher Scientific, Fisher Science, catalog number: S25287A )
Molybdic acid (H2MoO4) (Sigma-Aldrich, catalog number: 232084-100G )
Hoagland’s nutrient solution
High N (15 mM NO3-) Hoagland’s solution (see Recipes)
Low N (1.5 mM NO3-) Hoagland’s solution (see Recipes)
Micronutrient stock solution (1 L) (see Recipes)
30% ethanol (C2H6O) (commercial grade from any brand) (see Recipes)
2.5 g/L Fungicide solution Captan® (Bonide Products Inc.) (see Recipes)
Equipment
2 L capacity beakers (each beaker holds 8-10 paper rolls) (Coring, Pyrex® Griffin Beakers, catalog number: 10000-2L )
50 ml capacity beakers (Pyrex®, Griffin Beakers), for sterilizing and washing seeds (Sigma-Aldrich, catalog number: CLS100050 )
Plant growth chamber (Conviron, model: PGC FLEX )
Cold room (Rheem Puffer Hubbard environmental chamber) or refrigerator
Autoclave (PRIMUS Sterilizer , model: PSS5 )
Sensitive balance (Ohaus, model: AdventurerTM AR0640 )
Note: This product has been discontinued.
Flatbed scanner (Epson, model: Expression 10000 XL , or any other brand)
Computer with flash drive and Windows operating system
Fisher ScientificTM IsotempTM general purpose heating and drying oven (Thermo Fisher Scientific, Fisher Scientific, model: 15-103-0503) or any constant temperature oven/dryer
Ruler or yardstick (Acme Westcott 15728 36” Aluminum Yard Stick)
Software
WinRhizo (Regent Instruments, model: WinRhizo Pro 2009) or ARIA (Automatic Root Image Analysis) (Pace et al., 2014)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Abdel-Ghani, A. H., Sanchez, D. L., Kumar, B. and Lubberstedt, T. (2016). Paper Roll Culture and Assessment of Maize Root Parameters. Bio-protocol 6(18): e1926. DOI: 10.21769/BioProtoc.1926.
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Category
Plant Science > Plant developmental biology > Morphogenesis
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1,927 | https://bio-protocol.org/exchange/protocoldetail?id=1927&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia
Anupam Jhingran
SK Shinji Kasahara
TH Tobias M Hohl
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1927 Views: 15857
Reviewed by: Shanie Saghafian-Hedengren
Original Research Article:
The authors used this protocol in Feb 2015
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Abstract
Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.
Keywords: Fungus Microscopy Immunity Lung Reporter
Materials and Reagents
Cell strainers - 40 μm and 100 μm
Syringes
Injector (Modified precision dispensing tips) (Nordson, catalog number: 7018166 )
15 ml tubes
96-well plate, U-bottom
Mice: C57BL/6 (Jackson Laboratories, catalog number: 000664 )
Fungal strains (Af293 and Af293-DsRed) (Jhingran et al., 2012)
FATAL-plus solution (Vortech Pharmaceuticals Ltd, National Drug Code: 0298-9373-68 )
BD BBLTM Sabouraud dextrose agar (Emmons), slants (pH 6.9) (100/sp) (BD, catalog number: 221827 )
PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
Tween 20
Tris base
NaHCO3
Na2CO3
Biotin-XX, SSE (6-((6-((biotinoyl)amino)hexanoyl)amino) hexanoic acid, sulfosuccinimidyl ester, sodium salt) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: B6352 )
DMSO
Streptavidin, Alexa Fluor® 633 conjugate (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S-21375 )
IsothesiaTM, Isoflurane (Henry Schein Animal Health, SKU number: 029405 )
RBC lysis buffer (Biolegend, catalog number: 420301 )
DNase I (Roche Diagnostics, catalog number: 10104159001 )
Collagenase type IV (Worthington Biochemical, catalog number: LS004189 )
EDTA
Mouse BD Fc BlockTM (BD, BD PharmingenTM, catalog number: 553142 )
Antibodies (listed in Procedure section)
PBS Tween (0.025%) (see Recipes)
Tris-chloride (1 M, pH 8) (see Recipes)
Sodium bicarbonate buffer (0.1 M, pH 9.5) (see Recipes)
Lung digestion buffer (for each lung) (see Recipes)
FACS buffer (see Recipes)
Equipment
Hemocytometer
Microscope
Centrifuge
Plexiglass stand
Dissection scissors and forceps
Bronchoalveolar lavage fluid (BALF) catheters (Exel International, catalog number: 26738 )
4-way large bore (lipid resistant) stopcock with rotating male luer lock adapter (Baxter, catalog number: 2C6204 )
gentleMACS tissue dissociator, MACSMix tube rotator and C tubes (Miltenyi Biotech)
Automated cell counter or hemocytometer
Flow cytometer capable of analyzing at least 7 fluorescent parameters (such as BD LSRII). The 532 or 561 nm (but not 488 nm) laser excites DsRed fluorescence and the 633 nm laser excites Alexa Fluor 633 fluorescence.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Jhingran, A., Kasahara, S. and Hohl, T. M. (2016). Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia . Bio-protocol 6(18): e1927. DOI: 10.21769/BioProtoc.1927.
Heung, L. J., Jhingran, A. and Hohl, T. M. (2015). Deploying FLAREs to visualize functional outcomes of host-pathogen encounters. PLoS Pathog 11(7): e1004912.
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Category
Immunology > Immune cell staining > Flow cytometry
Microbiology > in vivo model > Fungi
Immunology > Host defense > Murine
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1,928 | https://bio-protocol.org/exchange/protocoldetail?id=1928&type=0 | # Bio-Protocol Content
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Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803
Simab Kanwal
Aran Incharoensakdi
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1928 Views: 11380
Edited by: Maria Sinetova
Reviewed by: Seda EkiciVinai Chittezham Thomas
Original Research Article:
The authors used this protocol in Jan 2015
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Jan 2015
Abstract
GABA (γ-amino butyric acid) is a biologically active four carbon non-protein amino acid found in prokaryotes and eukaryotes. Glutamate is a five carbon α-amino acid which can be converted to GABA catalyzed by the enzyme glutamate decarboxylase. In this protocol, we describe the procedure for extraction and quantification of GABA and glutamate from cells of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Apart from Synechocystis, this protocol has already been successfully tested for the cyanobacterium Nostoc punctiforme and for the green alga Tetraspora sp. CU2551. The protocol can also be used for the analyses of other primary amino acids. We have successfully employed this protocol in our studies of GABA and glutamate analyses in Synechocystis (Kanwal and Incharoensakdi, 2016).
Keywords: GABA assay Glutamate assay Amino acid analysis Cyanobacteria Synechocystis
Background
Amino acid analysis has been done previously by employing a number of methods that include the detection using thin layer chromatography or ion exchange separation coupled with post-column derivatization with ninhydrin method or also using HPLC by fluorescent detection. However, several challenges are encountered in those detection methods such as poor sensitivity, degradation of derivatives, formation of multiple derivatives and longer retention times causing higher solvent consumption. Furthermore, the UV detection methods alone could not be used owing to optically inactive nature of amino acids.
Here, we describe the detailed procedure for extraction and analyses of amino acids such as GABA and glutamate in Synechocystis. The method used for amino acids separation and detection is high performance reversed-phase liquid chromatography followed by UV detection of pre-column OPA (o-phthaldehyde) derivatization of amino acids adopted from Henderson et al. (2006). This method offers several advantages over previous methods by improving the sensitivity of detection. Reversed-phase chromatography allows faster separation of amino acids, whereas UV detection offers improved sensitivity. OPA derivatization of amino acids results in stable derivatives that make the UV detection of amino acids possible. The method also offers the detection and quantification of amino acids within 26 min, which consequently reduces solvent consumption.
Materials and Reagents
50 ml polypropylene tubes (Corning®, CentristarTM, Mexico)
1.5 ml Eppendorf tubes (Hycon plastics, catalog number: HCE-1004-MIC )
200 µl conical inserts with polymer feet (Borosilicate glass) (JG Finneran, USA)
Amber wide-opening vial with screw cap (Borosilicate glass) (JG Finneran, USA)
0.2 µm Millipore filter (Nylon or cellulose filter membrane) (Sartorius Stedim biotech, Germany)
Synechocystis cells (fresh or frozen at -20 °C)
BG11 growth medium
Deionized water
Methanol, AR grade (RCI Labscan, Thailand)
Ethanol, AR grade (QRec, New Zealand)
Chloroform (RCI Labscan, Thailand)
Hydrochloric acid (HCl) (QRec, New Zealand)
NaH2PO4 (Carlo Erba Reagents, Chaussée du Vexin, Val de Reuil, France)
NaOH (Merck, Germany)
UP water, HPLC grade (TEDIA, OH, USA)
Acetonitrile CAN, HPLC grade (Burdick and Jackson, Korea)
Methanol (MeOH), HPLC grade (Burdick and Jackson, Korea)
Standards of glutamate and GABA, > 99% purity (Sigma-Aldrich)
0.4 M borate buffer, pH 10.2 (Agilent Technologies, catalog number: 5061-3339 )
O-phthaldehyde reagent (OPA) (Agilent Technologies, catalog number: 5061-3335 )
BG11 growth medium (see Recipes)
HPLC solvent A (see Recipes)
HPLC solvent B (see Recipes)
OPA reagent (see Recipes)
Equipment
Pyrex® 250 ml conical flasks (Corning, Pyrex®, catalog number: 4980 )
Shaker (Eppendorf, model: InnovaTM 2100 )
Tabletop centrifuge (Hettich Lab Technology, model: MIKRO220/220R )
Vortex mixer (Scientific Industries, model: SI-0236 )
Ultrasonic laboratory homogenizer (Sonuplus UW2200)
Centrivap concentrator (Labconco, USA)
Shimadzu prominence ultra-fast liquid chromatography system (Shimadzu Scientific Instruments)
Shimadzu prominence autosampler (Shimadzu Scientific Instruments, model: SIL-21A HT )
Degasser (Shimadzu Scientific Instruments, model: DGU-20A5 )
UV-vis detector (Shimadzu Scientific Instruments, model: SPD-20A )
Reverse phase column (ZORBAX Eclipse AAA analytical column, 4.6 x 150 mm, 5.0 μm) (Agilent Technologies, catalog number: 93400-902 )
Guard column (ZORBAX Eclipse AAA analytical guard column, 4.6 x 12.5 mm, 5.0 μm) (Agilent Technologies, catalog number: 820950-931 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kanwal, S. and Incharoensakdi, A. (2016). Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803. Bio-protocol 6(18): e1928. DOI: 10.21769/BioProtoc.1928.
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Category
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Amino acid
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1,929 | https://bio-protocol.org/exchange/protocoldetail?id=1929&type=0 | # Bio-Protocol Content
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Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains
Konstantin Kulichikhin
SM Shalini Mukherjee
BA Belay T. Ayele
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1929 Views: 13210
Edited by: Samik Bhattacharya
Original Research Article:
The authors used this protocol in Nov 2015
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Abstract
Starch biosynthesis in plants involves a network of enzymes of which adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27), and soluble and granule bound starch synthases (SSS and GBSS, E.C. 2.4.1.21) play central roles. Here, we outline the protocol for extraction and assay of these enzymes in developing grains of wheat (Triticum aestivum L.). The principle of the assays outlined is based on a coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be measured easily by spectrophotometer. This protocol does not need expensive labelled chemicals and can be carried out using equipment commonly found in a biochemical laboratory. We applied this protocol to study the dynamics of AGPase, SSS and GBSS activity in developing wheat grains at different time points after anthesis.
Keywords: Enzyme Grain Starch synthesis Wheat
Background
Starch is a carbohydrate polymer made up of amylose, a linear glucan polymer composed of α-1,4-linked glucose molecules, and amylopectin, another glucan polymer composed of α-1,4-linked glucose molecules branched by α-1,6-glycosidic bonds. The enzyme adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27) catalyzes the first committed step of starch synthesis, converting glucose-1-phosphate and ATP to ADP-glucose and inorganic pyrophosphate (PPi). ADP-glucose is subsequently used by soluble starch synthases (SSS) and granule bound starch synthases (GBSS) (E.C. 2.4.1.21), and starch branching enzymes to elongate and branch the glucan chains of the starch granule.
Initially, AGPase and starch synthase assays were carried out using 14C- and 32P-labelled ADP-glucose (Ghosh and Price, 1966; Vos-Scheperkeuter et al., 1986), which requires the use of expensive chemicals as well as specialized equipment to work with labelled compounds. Here, we outline the method adopted and applied for extracting and assaying AGPase, SSS and GBSS activity in developing wheat (Triticum aestivum L.) grains (Mukherjee et al., 2015). Our protocol is based on the methods reported previously by Nakamura et al. (1989) for measuring AGPase and SSS activities in the endosperm of developing rice grains, and by Schaffer and Petreikov (1997) for measuring the activities of SSS and GBSS in tomato fruits. The method is based on coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be easily measured by spectrophotometers. The protocol can be carried out using the equipment commonly found in biochemical laboratories and does not require the use of labeled compounds.
Because this protocol is an adoption of the methods developed for different tissues of other plant species (endosperm of rice grain and tomato fruit), the reaction mixture composition for all enzymes was optimized for reaction buffer pH and substrate concentration so that enzyme activity was within the linear phase with respect to incubation time and protein concentration. The amounts of enzyme preparation added (PGM, pyruvate kinase, hexokinase, G6PDH) have been adjusted to achieve completion of coupling reaction in expected time frame. This protocol can be used for studying the activities of AGPase, SSS and GBSS in other tissues of wheat as well as in different tissues of other plant species; however, optimization of reaction buffer pH and substrate concentrations is required.
The principle of AGPase assay is presented in Figure 1. AGPase present in plant tissue extract catalyzes the conversion of ADP-glucose and PPi into glucose-1-phosphate and ATP. After the first reaction is stopped by boiling, phosphoglucomutase (PGM) is added to the reaction mixture; PGM converts quantitatively glucose-1-phosphate into glucose-6-phosphate. Subsequently, glucose-6-phosphate dehydrogenase (G6PDH) is added; G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid. At the same time, NADP is converted into NADPH and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose degraded by AGPase activity.
Figure 1. Principle for ADP-glucose pyrophosphorylase (AGPase) assay
The principle of SSS/GBSS assay is presented in Figure 2. Starch synthases present in plant tissue extract (SSS) or in starch granule suspension (GBSS) catalyze the conversion of ADP-glucose into ADP coupled with the elongation of amylopectin primer for one glucose residue. Pyruvate kinase added to the reaction mixture after the first reaction is stopped by boiling converts quantitatively ADP into ATP in the presence of PEP. Subsequently, addition of glucose together with hexokinase leads to the conversion of ATP into glucose-6-phosphate. In the next step of the assay, G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid; NADP is converted into NADPH at the same time and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose (at the first step of the analysis) degraded by SSS/GBSS activity.
Figure 2. Principle for soluble and granule bound starch synthases (SSS and GBSS) assay
Materials and Reagents
Chemicals
Common chemicals, buffers and kits
Protein assay kit (Bio-Rad Laboratories, catalog number: 5000002 )
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP310 )
Sodium hydroxide (NaOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP359 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
Ethylenediaminetetraacetic acid (EDTA), disodium salt dihydrate (Na2EDTA·2H2O) (Sigma-Aldrich, catalog number: E1644 )
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Polyvinylpyrrolidone (average molecular weight 10,000) (Sigma-Aldrich, catalog number: PVP10 )
Potassium chloride (KCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP366 )
Tetrasodium pyrophosphate (PPi) decahydrate (Na4P2O7·10H2O) (Sigma-Aldrich, catalog number: S6422 )
Substrates and cofactors
Adenosine-5’-diphosphoglucose (ADP-glucose), disodium salt (Sigma-Aldrich, catalog number: A0627 )
Amylopectin from maize (Sigma-Aldrich, catalog number: 10120 )
β-Nicotinamide adenine dinucleotide phosphate (NADP) sodium salt hydrate (Sigma-Aldrich, catalog number: N0505 )
Phospho(enol)pyruvic acid (PEP) monosodium salt hydrate (Sigma-Aldrich, catalog number: P0564 )
Enzymes
Hexokinase (E.C. 2.7.1.1) from Saccharomyces cerevisiae (Sigma-Aldrich, catalog number: H5000 )
Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) from Saccharomyces cerevisiae (Sigma-Aldrich, catalog number: G7877 )
Phosphoglucomutase (PGM, E.C. 5.4.2.2) from rabbit muscle (Sigma-Aldrich, catalog number: P3397 )
Pyruvate kinase (E. C. 2.7.1.40) from rabbit muscle (Sigma-Aldrich, catalog number: P1506 )
Stock solutions (see Recipes)
20 mM ADP-glucose
10 mM NADP
20 mM PEP
Enzymatic activities reaction mixtures
Extraction buffer (see Recipes, section 1)
AGPase activity reaction mixture (see Recipes, section 3a)
Starch synthase activity reaction mixture (see Recipes, section 3b)
Solution 1 for starch synthase activity assay (Pyruvate kinase reaction mixture) (see Recipes, section 3c)
Solution 2 for starch synthase activity assay (Glucose-6-phosphate dehydrogenase reaction mixture) (see Recipes, section 3d)
Plant material
Grains of common wheat (Triticum aestivum L.)
Other materials
Eppendorf tubes (1.5 and 2.0 ml)
Falcon tubes (15 ml)
Liquid nitrogen
Microcentrifuge tube locker (for example, Sigma-Aldrich, catalog number: Z708372 , or Sorenson Bioscience MCT LidLockTM, catalog number: 11870 )
Note: Product Z708372 has been discontinued.
Miracloth (Merck Millipore, catalog number: 475855 )
Equipment
Porcelain mortar and pestle
Analytical balance (capacity - 100 g or higher, resolution - 0.001 g or better)
Digital heat block or water bath suitable for 30 °C and digital heat block or water bath suitable for 100 °C
Note: For some steps of the analysis, immediate transfer from 30 °C to 100 °C is necessary. This is why two heating devices are necessary simultaneously for the protocol.
Refrigerating ultracentrifuge with appropriate rotor suitable for spinning of 15 ml Falcon tube and 1.5-2 ml Eppendorf tubes at up to 12,000 x g
Ultraviolet (UV)/Visible spectrophotometer equipped with heated cell holder suitable for maintaining cuvette temperature at 30 °C. We used GE Healthcare/Amersham Bioscience Ultrospec 3100 Pro UV/Visible spectrophotometer (Biochrom Ltd, Cambridge, UK).
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kulichikhin, K., Mukherjee, S. and Ayele, B. T. (2016). Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains . Bio-protocol 6(18): e1929. DOI: 10.21769/BioProtoc.1929.
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Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Isolation and purification
Plant Science > Plant biochemistry > Protein
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193 | https://bio-protocol.org/exchange/protocoldetail?id=193&type=0 | # Bio-Protocol Content
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Synchronize Human Embryonic Stem Cells at Different Cell Cycle Stage
Hui Zhu
Published: Vol 2, Iss 11, Jun 5, 2012
DOI: 10.21769/BioProtoc.193 Views: 20780
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Abstract
Pluripotency and the capability for self-renewal are essential characteristics of human embryonic stem cells (hESCs), which hold great potential as a cellular source for tissue replacement. Short cell cycle (15-16 h) compared to somatic cells is another property of hESCs. Efficient synchronization of hESCs at different cell cycle stages is important to elucidate the mechanistic link between cell cycle regulation and cell fate decision. This protocol describes how to establish synchronization of hESCs at different cell cycle stages.
Keywords: Cell cycle Synchronize Human embryonic stem cells
Materials and Reagents
Human embryonic stem cells (hESCs) Undifferentiated hESCs were cultured on irradiated mouse embryonic fibroblast (MEF) feeders in DMEM/F12 medium supplemented with 20% KnockOut serum replacement, 0.1 mM nonessential amino acids (NEAA), 1 mM GlutaMAXTM-1, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and 8 ng/ml recombinant human FGF2 (Peprotech, catalog number: 100-18 B).
Matrigel (BD Biosciences, catalog number: 354230 )
Nocodazole (Sigma-Aldrich, catalog number: M1404 )
Thymidine (Sigma-Aldrich, catalog number: T1895 )
Aphidicholine (Sigma-Aldrich, catalog number: A0781 )
Phosphate buffered saline (PBS)
Dimethyl sulfoxide (DMSO)
Nocodazole stock solution (see Recipes)
Thymidine stock solution (see Recipes)
Aphidicholine stock solution (see Recipes)
Equipment
Centrifuges (Eppendorf 5415D centrifuge)
Fluorescence activated cell sorter (FACS, FACScan machine, Stanford FACS facility)
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Stem Cell > Embryonic stem cell > Cell-based analysis
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1,930 | https://bio-protocol.org/exchange/protocoldetail?id=1930&type=0 | # Bio-Protocol Content
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59Fe Uptake Assays in Paracoccidioides Species
DK Daniel J. Kosman
EB Elisa Flávia Luiz Cardoso Bailão
MS Mirelle Garcia Silva-Bailão
CS Célia Maria de Almeida Soares
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1930 Views: 6866
Edited by: Arsalan Daudi
Reviewed by: Belen SanzSoazig Le Guyon
Original Research Article:
The authors used this protocol in Oct 2015
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Abstract
Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the presence of ascorbic acid, the reductive-independent 59Fe uptake route is investigated. On the other hand, in the absence of ascorbic acid, the reductive-dependent 59Fe uptake route is stimulated. Using this strategy for the human pathogenic fungus Paracoccidioides species, the results showed that iron uptake by Pb01 in the absence of ascorbic acid was low, unlike what was observed for Pb18. These results suggest that only in Pb18 the iron uptake pathway is coupled to a ferric reductase (Bailão et al., 2015). In this protocol, we describe how to perform 59Fe uptake assays in Paracoccidioides species.
Keywords: Reductive iron assimilation pathway Reductive-independent iron uptake route Ascorbic acid Quench buffer Gamma counter
Materials and Reagents
Conical tube
Wide-mouth screw-cap high-density polyethylene (scintillation) vials
13 x 100 mm test tubes
Petri dish
Filter type A/C 25 mm glass fiber filters from Pall Gelman
12 x 75 mm glass test tubes
Wooden applicator (a thin wooden stick that is used in this protocol to put the filters containing radioactive cells inside the test tubes that will be read by the G counter)
Disposable vacuum filtration system (TPP, catalog number: 99950 )
Paracoccidioides spp. cells
Culture media
Liquid Brain Heart Infusion Broth (BHI) (Sigma-Aldrich, catalog number: 53286 ) with 4% glucose
Liquid Synthetic Complete (SC medium) (see Recipes)
Deionized water
Yeast Nitrogen Base (YNB) without amino acids (BD, DifcoTM, catalog number: 291940 )
Glucose (VWR, catalog number: 101175P )
Amino acids (Sigma-Aldrich)
ADE, TRP, HIS, ARG, MET, TYR, ISOLEU, LYS, PHE, GLU. ACID, ASP. ACID, VAL, THR, SER, URA, LEU
Trypan blue solution, 0.4% for microscopy (Sigma-Aldrich, catalog number: 93595 )
Ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Succinic acid (Sigma-Aldrich, catalog number: 398055 )
Tris
NaOH
Analar glucose
MES buffer (Sigma-Aldrich, catalog number: M3671 )
Sodium citrate
59Fe (Perkin-Elmer, catalog number: NEZ037001MC )
1 M ascorbic acid (0.176 g in 1 ml sterilized water)
Nitric acid (HNO3)
Bathophenanthrolinedisulfonic acid (BPS) (Sigma-Aldrich, catalog number: B1375 )
Iron (III) chloride (Sigma-Aldrich, catalog number: 157740 )
SC complete media (see Recipes)
Amino acids solution (see Recipes)
Amino acid mix stock (see Recipes)
59Fe stock solutions (see Recipes)
10x quench buffer (see Recipes)
Citrate uptake buffer (see Recipes)
Acid-wash glassware treatment (see Recipes)
Equipment
Shaker
Hemocytometer
Microfine forceps
Glassware (erlenmeyers, bottle with blue cap, beakers, cylinders)
Magnetic stirrer
pH meter
Vortex
G counter (LKB Wallac Compu Gamma) with plastic g counting tube carriers
Vacuum filtration manifold (Merck Millipore, model: 1225 )
Source of vacuum, either standard lab pump or water aspirator
Autoclave
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kosman, D. J., Bailão, E. F. L. C., Silva-Bailão, M. G. and Soares, C. M. D. A. (2016). 59Fe Uptake Assays in Paracoccidioides Species. Bio-protocol 6(18): e1930. DOI: 10.21769/BioProtoc.1930.
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Category
Microbiology > Microbial metabolism > Other compound
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Ion
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1,931 | https://bio-protocol.org/exchange/protocoldetail?id=1931&type=0 | # Bio-Protocol Content
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Peer-reviewed
Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells
LC Loredana Cifaldi
FL Franco Locatelli
Doriana Fruci
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1931 Views: 6966
Edited by: HongLok Lung
Reviewed by: Benoit Chassaing
Original Research Article:
The authors used this protocol in Mar 2015
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Abstract
Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses the ligand, this protocol will allow to evaluate its interaction with the specific receptor. In particular, killer cell immunoglobulin (Ig)-like receptors (KIR) recognize their ligands (HLA class I molecules) through the direct contact with HLA class I heavy chain residues and amino acid residues of the bound peptide. This protocol will allow to test the effect of amino acid substitutions or other mutations on the binding of KIR to HLA class I. We used this protocol to depict the role of ERAP1, a key component of the MHC class I antigen processing, in regulating NK cell function by controlling the engagement of inhibitory receptors (Cifaldi et al., 2015).
Keywords: NK cells Killer cell immunoglobulin (Ig)-like receptors (KIR) HLA class I KIR funsion protein Tumor cells
Materials and Reagents
T25 Flasks (Corning, Falcon®, catalog numbers: 353108 )
T75 Flasks (Corning, Falcon®, catalog numbers: 353136 )
15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
96 well plates (Corning, catalog number: 3799 )
FACS tubes
Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO2)
RPMI 1640 (EuroClone, catalog number: ECM9106L )
Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
L-glutamine (EuroClone, catalog number: ECB3000D )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
Trypan-blue
KIR-Fc fusion proteins
Recombinant Human KIR2DL1/CD158a Fc chimera protein (R&D System, catalog number: 1844-KR-050 )
Recombinant Human KIR2DL3/CD158b2 Fc chimera protein (R&D System, catalog number: 2014-KR-050 )
Recombinant Human KIR3DL1 Fc chimera protein (R&D System, catalog number: 1225-KR-050 )
Goat anti-Human Molecular ProbesTM secondary antibody, RPE conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: H10104 )
Complete RPMI (see Recipes)
833 μM EDTA (see Recipes)
FACS buffer (see Recipes)
Equipment
Centrifuge, used with maximal acceleration and deceleration (Eppendorf, model: 5810R )
Incubator (5% CO2, 37 °C) (FormaTM Steri-CultTM CO2 Incubator-Thermo Fisher Scientific)
Flow cytometer, equipped with 2 lasers (8 detectors), interfaced with PC by using DIVA Software version 6.1.3 (BD, model: FACSCanto II )
Software
DIVA software version 6.1.3
FlowJo software
Procedure
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Category
Immunology > Immune cell function > Antigen-specific response
Cancer Biology > Tumor immunology > Cell biology assays
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1,932 | https://bio-protocol.org/exchange/protocoldetail?id=1932&type=0 | # Bio-Protocol Content
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Peptide Loading on MHC Class I Molecules of Tumor Cells
LC Loredana Cifaldi
FL Franco Locatelli
Doriana Fruci
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1932 Views: 10548
Edited by: HongLok Lung
Reviewed by: Benoit Chassaing
Original Research Article:
The authors used this protocol in Mar 2015
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Abstract
MHC class I molecules present peptides to cytotoxic T cells allowing the immune system to scan for intracellular pathogens and mutated proteins. The generation of antigenic peptides is a multistep process that ends in the endoplasmic reticulum (ER). Only peptides with the right length and sequence will bind nascent MHC class I molecules in the ER. This protocol allows for detachment of the endogenous peptides bound to MHC class I molecules by preserving them for the binding of high affinity synthetic peptides. The complete dissociation of endogenous peptides by mild acid treatment as well as the binding of synthetic peptides to MHC class I molecules will be evaluated measuring HLA class I molecules express on the cell surface by flow cytometry. The mouse antibody W6/32 which recognizes β2m associated HLA-A, -B, -C, -E and -G heavy chains is suitable for this propose. Any tumor cell line that expresses surface HLA class I molecules is suitable for the assay. Another important aspect is to know the HLA class I typing of tumor cell line to allow selection of the known high affinity peptides.
Keywords: MHC class I High affinity peptides Acid treatment MHC class I surface expression Tumor cells
Materials and Reagents
T25 Flasks (Corning, Falcon®, catalog numbers: 353108 )
T75 Flasks (Corning, Falcon®, catalog numbers: 353136 )
15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
6 well plates (Corning, Falcon®, catalog number: 353046 )
1.5 ml Safe-Lock tubes (Eppendorf, catalog number: 0030120086 )
96 well plates (Corning, catalog number: 3799 )
FACS tubes
Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO2)
RPMI 1640 (Euroclone, catalog number: ECM9106L )
Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, Gibco®, catalog number: 10270-106 )
Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
L-glutamine (EuroClone, catalog number: ECB3000D )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
Trypan-Blue
Na2HPO4 (Sigma-Aldrich, catalog number: S5136 )
Citric acid monohydrate (Sigma-Aldrich, catalog number: 251275 )
mAb W6/32
Beta 2-microglobulin (Sigma-Aldrich, catalog number: M4890 )
Peptides (lyophilized, resuspended in DMSO at 10 mM) (Anaspec)
Goat F(ab’)2 Fragment anti-mouse IgG (H+L)-FITC (Beckman Coulter, catalog number: PN IM0819 )
Complete RPMI (see Recipes)
833 μM EDTA (see Recipes)
FACS buffer (see Recipes)
Acid buffer (see Recipes)
Equipment
Centrifuge, used with maximal acceleration and deceleration (Eppendorf, model: 5810R )
Incubator (5% CO2, 37 °C) (FormaTM Steri-CultTM CO2 Incubator-Thermo Fisher Scientific)
Flow cytometer equipped with 2 lasers (8 detectors), interfaced with PC using DIVA Software version 6.1.3 (BD, model: FACSCanto II )
Software
DIVA software version 6.1.3
FlowJo software
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cifaldi, L., Locatelli, F. and Fruci, D. (2016). Peptide Loading on MHC Class I Molecules of Tumor Cells. Bio-protocol 6(18): e1932. DOI: 10.21769/BioProtoc.1932.
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Category
Immunology > Immune cell function > Antigen-specific response
Cancer Biology > Tumor immunology > Cell biology assays
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1,933 | https://bio-protocol.org/exchange/protocoldetail?id=1933&type=0 | # Bio-Protocol Content
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Peer-reviewed
Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test
Jeremy Ferrier*
Fabien Marchand*
David Balayssac
*Contributed equally to this work
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1933 Views: 26980
Edited by: Oneil G. Bhalala
Reviewed by: Antoine de Morree
Original Research Article:
The authors used this protocol in Dec 2015
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Abstract
Chronic pain is one of the most debilitating conditions, affecting one out of five people worldwide. Preclinical models in rodents represent a valuable tool for the study of pathophysiological mechanisms and the discovery of new analgesic drugs. However, pain evaluation in rodents is rather challenging. Altered response to mechanical or thermal stimuli is commonly used as behavioral outcome to measure pain sensitivity. This protocol introduces a method for assessing static mechanical pain hypersensitivity in rats using an electronic von Frey (VF) test. The electronic VF is an evolution of the manual VF hairs previously described by Kim and Chung (1991). In this previous procedure, 6 calibrated nylon filaments (diameters of 0.13, 0.23, 0.31, 0.48, 0.52, and 0.59 mm, respectively) are perpendicularly applied to the plantar surface of the rat hind paw, to deliver the following defined pressures: 5.89, 9.81, 27.0, 74.4, 124, and 205 mN. Each application has to be repeated several times for each filament to determine the mechanical threshold. Comparatively, the electronic VF is relatively easy-to-use and particularly suited for pharmacological studies with precise time-points. The electronic VF can be used as a behavioural read-out for a wide range of models, including inflammatory and neuropathic pain. The following protocol was originally published in Ferrier et al. (2013), Grégoire et al. (2014) and in Ferrier et al. (2015).
Keywords: Electronic von frey Allodynia Rat Neuropathy Pain
Materials and Reagents
Plastic tip (Harvard Apparatus, catalog number: 76-0488 )
Note: The tip doesn’t need to be changed between rats and between sessions as long as it is not damaged or dirty beyond cleaning.
Rats
Note: To reach a sufficient statistical power, use a minimum of 8 rats per group. Use preferentially Sprague-Dawley male rats, weighing 200 to 400 g (6 to 12 weeks old), since this protocol has only been validated on this strain of rats. However, there are no obvious reasons that this protocol won’t be suitable for others strains of rats.
Cleaning solution
Note: Within one session, the boxes are carefully cleaned with water to remove the odor of the previous animal. At the end of the session, clean the boxes using 70% ethanol.
Oxaliplatin (6 mg/kg, dissolved in 5% glucose)
Equipment
Apparatus (either home-made or commercially available at Harvard Apparatus, catalog number: 76-0886/87 )
Note: The apparatus consists of an elevated horizontal wire mesh stand (Figure 1). The dimensions of the Plexiglas boxes set on top of the wire grid are 17 x 11 x 13 cm. The boxes are covered with perforated lids. Frosted or opaque separators can be used to diminish interactions between rats.
Figure 1. Apparatus for the electronic von Frey test
Electronic von Frey test (BIO-EVF4, Bioseb®, France)
Note: The apparatus consists of a portable force transducer fitted with a plastic tip (Figure 2). When applying a pressure on the tip, the maximal force applied (in grams) is automatically recorded by the electronic device and displayed on the screen. The maximal pressure (cut-off) will be reached when the paw of the animal is lifted by the plastic tip. Between two stimulations (separated by at least 3 min), the force value is set to zero simply by pushing a foot pedal.
Figure 2. Electronic von Frey test device
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Ferrier, J., Marchand, F. and Balayssac, D. (2016). Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test. Bio-protocol 6(18): e1933. DOI: 10.21769/BioProtoc.1933.
Ferrier, J., Bayet-Robert, M., Dalmann, R., El Guerrab, A., Aissouni, Y., Graveron-Demilly, D., Chalus, M., Pinguet, J., Eschalier, A., Richard, D., Daulhac, L., Marchand, F. and Balayssac, D. (2015). Cholinergic neurotransmission in the posterior insular cortex is altered in preclinical models of neuropathic pain: key role of muscarinic M2 receptors in donepezil-induced antinociception. J Neurosci 35(50): 16418-16430.
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Category
Neuroscience > Sensory and motor systems > Animal model
Neuroscience > Neuroanatomy and circuitry > Animal model
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1,934 | https://bio-protocol.org/exchange/protocoldetail?id=1934&type=0 | # Bio-Protocol Content
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Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts
KS Kenji Sugiyama
KN Kyosuke Nagata
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1934 Views: 9946
Edited by: Yannick Debing
Reviewed by: Vasudevan AchuthanModesto Redrejo-Rodriguez
Original Research Article:
The authors used this protocol in Nov 2015
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Abstract
Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we describe detailed methods for successive procedures for identification and purification of IREF-2, including large-scale suspension culture of HeLa S3 cells, preparation of NEs and separation of IREF-2 by sequential column chromatography steps. This protocol can be modified and used for purification and identification of the other unknown nuclear protein(s) of your interest.
Keywords: Nuclear Extracts Purification and identification Influenza Host factor IREF-2
Background
The influenza A virus genome is composed of 8 segmented- and single stranded-RNA (vRNA). Its transcription and replication are catalyzed by virus-encoded RNA-dependent RNA polymerase (RdRP). Several lines of evidence suggest that certain host-derived factors regulate the viral RNA syntheses (Nagata et al., 2008). Recently, a variety of host-derived proteins have been reported as regulator candidates related to the viral RNA syntheses by interactome analyses and genome wide RNAi screening studies. Among them, however, some false-positive interactors and factors involved indirectly in the viral RNA synthesis seem to be included. Instead, to identify reliable and important host factor(s) which play a direct role in the viral RNA synthesis process, we utilized a biochemical complementation assay system. In this system, the viral vRNA replication reaction occurring efficiently in infected cell nuclei is dissected and reconstituted in vitro using viral factors essential for the viral RNA replication such as viral RdRP derived from detergent-solubilized virion particle and model viral genomic RNA templates and uninfected nuclear extracts (NEs).
Recently, we have reported that an efficient vRNA replication is reproduced in vitro with viral factors and a crude fraction of NEs (Sugiyama et al., 2015). This stimulatory activity presented in NEs, designated IREF-2 that allows robust vRNA replication, was further fractionated and purified by sequential column chromatography. Finally, two host-derived paralogous proteins, pp32 (accession number NP_006296) and APRIL (accession number NP_006392), were identified by MS spectrometry analyses. Following experiments using recombinant pp32 and APRIL, and also in vivo analyses using siRNA confirmed that these host proteins are authentic proteins responsible for the IREF-2 activity (Sugiyama et al., 2015).
Materials and Reagents
Tissue culture dish
Centrifuge tubes [e.g., for F0650 rotor (Beckman Coulter, catalog number: 363075 )]
Conical centrifuge tubes (polypropylene, 50 ml) (e.g., Corning, catalog number: 430829 )
Conical centrifuge tubes (polypropylene, 15 ml) (e.g., Corning, catalog number: 430791 )
Dialysis membrane tube (Cellulose membrane, 14,000 dalton molecular weight cut off), (EIDIA, catalog number: UC27-32-100 )
Bottle top filter (Merck Millipore Corporation, catalog number: SCGPS01RE )
Microcentrifuge tubes (polypropylene, 1.5 ml size and 0.6 ml size) (e.g., Eppendorf)
HeLa S3 cell [National Institutes of Biomedical Innovation, Japanese Collection of Research Bioresources (JCRB), catalog number: JCRB0713 ]
Minimum essential medium Eagle (MEM) (Sigma-Aldrich, catalog number: M4655 )
Fatal bovine serum (FBS) (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 26140-079 )
S-MEM [Sigma-Aldrich, catalog number: M4767 (production ceased, see Note 1). Alternatively, see also Sigma-Aldrich, catalog number: M8167 , or similar products from other companies]
Bovine serum (CS) (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 16170-078 )
Ultra-pure water (Milli-Q)
Phosphate buffered saline (PBS)
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) buffer
Sodium hydroxide (NaOH)
Hydrochloric acid (HCl)
Sodium chloride (NaCl)
Potassium chloride (KCl)
Magnesium chloride hexahydrate (MgCl2)
Dithiothreitol (DTT)
Trypsin-EDTA (for cell trypsinization)
Glycerol
di-Sodium dihydrogen ethylenediamine tetraacetate dihydrate (EDTA)
Phenylmethylsulfonyl fluoride (PMSF)
Dimethyl sulfoxide (DMSO)
Liquid N2
Whatman® cation exchange cellulose (P11) (Sigma-Aldrich, catalog number: 4071010 )
Ammonium sulfate [(NH4)2SO4]
Silver staining kit (e.g., Cosmo Bio, catalog number: DCB-423413 )
Silver staining kit (glutaraldehyde-free for MS analysis) (e.g., Wako Pure Chemical industries, catalog number: 299-58901 )
Buffer A (see Recipes)
Buffer C (see Recipes)
Buffer D (see Recipes)
Buffer HG (see Recipes)
Buffer HGK (see Recipes)
Buffer HGN1M (see Recipes)
Buffer HGN2M (see Recipes)
Equipment
Cell culture environments and equipment
Clean bench
CO2 incubator (37 °C for adherent cultivation)
Microscope (phase-contrast)
Hemocytometer
Spinner flasks (small size for ~500 ml and large size for 8 L)
Magnetic stirrer (Slow speed; enough size for spinner flask)
37 °C incubator (enough size for spinner flask and magnetic stirrer)
Autoclave (enough size for spinner flask)
Large scale centrifuge (e.g., Beckman Coulter, model: Avanti® J-26XP )
Large scale centrifuge rotor (Beckman Coulter, model: JLA-10.500 )
Centrifuge bottles (500 ml-size for JLA-10.500 rotor) (e.g., Beckman Coulter, catalog number: 361691 )
Benchtop high speed centrifuge (e.g., Beckman Coulter, model: Allegra® 64R )
High-speed centrifuge rotor [e.g., F0650 (Beckman Coulter, catalog number: 364610 )]
Grass Dounce homogenizer with pestle, 15 ml (Capitol Scientific, Wheaton®, catalog number: 357544 )
Grass Dounce homogenizer with pestle, 40 ml (Capitol Scientific, Wheaton®, catalog number: 357546 )
Refrigerator (for dialysis)
Deep freezer (-80 °C)
pH meter
Grass bottles (Pyrex®, dry heat-sterilizable)
Tabletop centrifuge
SDS-PAGE apparatus
Column chromatography equipment (see Note 1)
Fast protein liquid chromatography (FPLC) workstation (Bio-Rad Laboratories, model: BioLogic HR )
Chromato chamber (4 °C)
Column housing (e.g., Bio-Rad Laboratories, Econo Column®, catalog number: 7371512 )
Flow adaptor (e.g., Bio-Rad Laboratories, catalog number: 7380016 )
Anion-exchanger, UNOTM Q1R (Bio-Rad Laboratories, catalog number: 7200011 )
Hydrophobic column, SOURCETM 15PHE PE 4.6/100 (GE Healthcare, catalog number: 17-5186-01 )
Cation-exchanger, UNOTM S1 (Bio-Rad Laboratories, catalog number: 7200021 )
Anion-exchanger, Mono Q® HR 5/5 [GE Healthcare, model: Mono Q® HR 5/5 (production ceased). Alternatively, Mono Q® 5/50 GL (GE Healthcare, model: 17-5166-01 ) or similar products from other companies can be usable]
Micro-Purification SMART system [GE Healthcare, Pharmacia Biotech, model: SMART® system (production ceased)]
Gel filtration column (GE Healthcare, Pharmacia Biotech, model: Superdex® 75 PC 3.2/30 )
Note: This product has been discontinued.
Razor blade
Procedure
Large scale culture of HeLa S3 cells (see Note 2)
Seed HeLa S3 cells on tissue culture dish(es) in MEM + 10% FBS medium.
Incubate at 37 °C in 5% CO2 to grow until total cell number reaches to ≥ 1 x 107. Keep the density of adherent cells within 30-80% confluence. If necessary, passage all of the growing cells by increasing size and/or numbers of culture dish(es).
Trypsinize the cells, resuspend in S-MEM + 5% calf serum (CS), and count the number of cells.
Adjust the cell density to ~2 x 105 cells/ml by adding S-MEM + 5% CS.
Place the cell suspension into a 500 ml-size spinner flask (sterilized), and incubate at 37 °C with gentle stirring by a magnetic stirrer (60-80 rotations/min).
Monitor the cell growth by daily counting the number of cells, and add fresh medium (S-MEM + 5% CS) to maintain cells at 3-6 x 105 cells/ml. When the volume of cell culture reaches to 500 ml, transfer the culture to a large-scale spinner flask (8 L-size) and maintain incubation at 37 °C.
When the cell culture reaches maximum volume (e.g., 8 L of 6 x 105 cells/ml; approximately 5 x 109 of total cells), proceed to the nuclear extracts preparation steps (see Note 3).
Nuclear Extracts (NEs) preparation from HeLa S3 cells (see Note 4)
Harvest the cultured HeLa S3 cells by centrifugation (at 1,500 x g, 4 °C for 10 min).
Suspend the cell pellet in PBS, and collect by centrifugation.
Suspend the cells in ~5 packed cell volumes (PCV) (i.e., pellet mass of the collected cells) of ice-cold buffer A, and store on ice for 10 min (see Note 5).
Collect by centrifugation at 1,500 x g for 10 min at 4 °C.
Suspend the cells in 2 PCV of the buffer A and allow cells to swell on ice for 10 min.
Lyse the cells by 10-15 strokes of a Dounce homogenizer with “type B (tight)” pestle.
Check for the cell lysis under a microscope (Figure 1). If the homogenization appears to be insufficient, homogenize additionally and check the lysis again.
Figure 1. Microscopic view of the cell homogenate. Suspensions before and after homogenization (step B6) were observed at 200-fold magnification by phase-contrast microscopy. A. Cells suspended in PBS (step B2); B. Swollen cells in a hypotonic “buffer A” before homogenization (step B5); C. Homogenized cell suspension (step B6). Cytoplasmic debris and nuclei were observed.
Centrifuge the homogenate at 3,300 x g for 10 min at 4 °C, and remove the supernatant. The supernatant removed at this step can be saved as “S-100” cytoplasmic extracts (Dignam et al., 1983), and utilized for other purpose (Matsumoto et al., 1993).
Re-centrifuge the remaining pellet (i.e., nuclei) at 14,000 x g for 20 min at 4 °C and remove the residual supernatant completely.
Suspend the cells in an equal volume of packed nuclei (i.e., equal to pellet mass of the collected nuclei) with ice-cold buffer C (see Recipes and Note 6) and homogenate by 10 strokes of a Dounce homogenizer with “type B (tight)” pestle to ensure complete suspension.
Transfer the homogenate to a centrifuge tube and invert gently by rotating for 30 min at 4 °C.
Centrifuge the homogenate at 25,000 x g for 10 min at 4 °C.
Place the supernatant (NEs) in a dialysis membrane bag. The remaining pellet at this step can be frozen and stored at -80 °C, and utilized as a chromatin fraction (Simon and Felsenfeld, 1979; Schnitzler, 2001).
Dialyze against ≥ 50 volumes of buffer D at 4 °C for 3 h. Change the buffer to fresh buffer D (≥ 50 volumes), and additionally dialyze at 4 °C for 3 h.
Transfer the dialysate (white-turbid NEs) to a centrifuge tube and centrifuge for 20 min at 25,000 x g by benchtop centrifuge.
Determine the protein concentration of NEs of the supernatant (see Note 7). Dispense aliquots into tubes (if desired), and freeze in liquid N2 and store at -80 °C until use (see Note 8).
Fractionation and purification of NEs by successive column chromatographies (see Figure 2 and Note 9). Herein, the procedures of the column chromatography are desired to purify and identify two host-derived factors, pp32 and APRIL (as known as ANP32A and B, respectively), termed as “influenza virus replication factor (IREF)-2” which stimulates influenza virus RNA replication process (Sugiyama et al., 2015).
Figure 2. Purification scheme of IREF-2 from NEs. Chromatographic behavior of IREF-2 appears to be highly acidic and hydrophilic.
Stepwise fractionation on weak cation-exchanger column
Prepare phosphocellulose (PC) column, as follows:
Suspend the powder of PC resin (Whatman® cation exchange cellulose, P11) by gentle swirling in ≥ 20 volumes of water and keep for 30~60 min.
Pour off supernatant, resuspend the resin in water, and repeat at least 8-10 times to remove any unsettled fine.
Suspend the resin in 0.2 M NaOH, allow to settle for 30 min.
Pour off supernatant and repeat until pH of supernatant is above 10.
Wash the resin with water until pH of supernatant is below 8.
Suspend the resin in 0.2 M HCl, allow to settle.
Pour off supernatant and repeat until pH of supernatant is below 3.
Wash the resin with water until pH of supernatant is about 5.
Equilibrate the resins in buffer HG. If the PC resin is to be stored for longer than a week, add 0.1% sodium azide and store at 4 °C (never freeze).
Suspend the PC resin and pour carefully into the column housing [approximately, 10 ml (bed volume) of the PC resin is enough for 10~15 ml of input NEs] and allow to settle. Then, attach the flow-adaptor carefully above the bed.
Set up the FPLC system and column, routinely as follows:
Wash the system (including a “sample loop”) with pure degassed water.
Connect the prepared PC column (10 ml bed volume) to the system without introducing air, and wash the system including the column with enough water. Ensure no leakage from the column during flow.
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
Wash the system with 100% solvent B (buffer HGK; 1 M KCl) and then equilibrate with 5% solvent B (i.e., 95% solvent A of buffer HG; 50 mM KCl).
Ensure the valve position to be “load”, and load the NEs (approximately, 12.5 ml in this protocol) into the sample loop.
Change the valve position to “inject”, and run the program to start stepwise fractionation on the PC column (10 ml bed volume), as follows:
Isocratic flow (0.15 ml/min) with 45 ml (4 volumes of the input NEs) of 5% solvent B (i.e., 95% solvent A; final 0.05 M KCl), collect the eluate (2 ml/fraction; total 23 fractions).
Change the valve position from “inject” to “load” to bypass the flow channel through the sample loop. If forget this, following salt-elution steps would be delayed by the volume of the sample loop.
Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 20% solvent B (i.e., 80% solvent A; final 0.2 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 50% solvent B (i.e., 50% solvent A; final 0.5 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
Isocratic flow (0.15 ml/min) with 40 ml (4 column volumes) of 100% solvent B (i.e., 0% solvent A; final 1 M KCl), collect the eluate (2 ml/fraction; total 20 fractions).
Make small aliquot(s) of each fractionated sample to avoid unnecessary freezing/thawing cycle in future, and freeze in liquid N2 and store at -80 °C.
Check the activity (i.e., stimulation activity for influenza virus RNA replication, termed as “IREF-2” in this protocol) involved in the PC-fractions by employing a cell-free viral RNA replication assay using small aliquot (see Figure 3 and Note 10).
Figure 3. Profile of the fractions from phosphocellulose column chromatography for IREF-2. Each stepwise fraction of NE from the phosphocellulose (PC) column chromatography was individually added to the cell-free viral RNA replication reaction (1.75 μg of each fraction/reaction), as follows; unbound fraction (i.e., flow-through) eluted with 0.05 M KCl buffer (lane 1), materials eluted with buffer containing 0.2 M KCl (lane 2), 0.5 M KCl (lane 3) and 1 M KCl (lane 4), respectively. For the detail of the cell-free viral RNA replication assay, see Note 10 and the original article (Sugiyama et al., 2015). Robust viral RNA product was detected (upper panel, lane 1), suggesting that certain activity stimulating a viral RNA replication reaction, termed as IREF-2, was present in the flow-through fraction from the PC column chromatography. Each fraction was also subjected to 14% SDS-PAGE (1 μg of each fraction/lane), and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. The molecular weight (kDa) positions are denoted on the right side of the lower panel. MWM: molecular weight marker.
Stepwise fractionation on a strong anion-exchanger column
Set up the FPLC system and pre-packed UNO Q1R column, routinely
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
Wash the column with 100% solvent B, and equilibrate with 5% solvent B (i.e., 95% solvent A).
Thaw the IREF-2 activity-positive fraction (total 9 ml in this protocol) of the PC column chromatography (i.e., the unbound “flow-through” fraction, see Figure 3) and centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load the supernatant into the sample loop and change the valve position to “inject”. Run the program to start stepwise fractionation on the UNO-Q column (1.3 ml column volume) as follows:
Isocratic flow (0.5 ml/min) with 18 ml (2 volumes of the input) of 5% solvent B (i.e., 95% solvent A; final 0.05 M KCl), collect the eluate (1 ml/fraction; total 18 fractions).
Change the valve position from “inject” to “load”.
Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 15% solvent B (i.e., 85% solvent A; final 0.15 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 30% solvent B (i.e., 70% solvent A; final 0.3 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 60% solvent B (i.e., 40% solvent A; final 0.6 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
Isocratic flow (0.5 ml/min) with 7.8 ml (6 column volumes) of 100% solvent B (i.e., 0% solvent A; final 1 M KCl), collect the eluate (1 ml/fraction; total 8 fractions).
Make small aliquot(s) of each stepwise-fractionated sample by the UNO-Q column, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10).
Gradient fractionation on a strong anion-exchanger column
Set up the FPLC system and the UNO Q1R column, routinely
Set degassed buffer HG as “solvent A” to inlet A and degassed buffer HGK as “solvent B” to inlet B, respectively.
Wash the column with 100% solvent B, and equilibrate with 25% solvent B (i.e., 75% solvent A).
Thaw the IREF-2-active fractions of the stepwise fractionated sample on the UNO-Q column chromatography (= total 8 ml), the eluate with 0.6 M KCl [i.e., mixture of 60% of buffer HGK (as solvent B) and 40% of buffer HG (as solvent A)], and dilute with 1.5 volume of buffer HG to decrease the salt concentration in the sample (finally, 20 ml as an input volume). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load the supernatant into the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the UNO-Q column (1.3 ml column volume) as follows:
Isocratic flow (0.5 ml/min) with 30 ml (~1.5 volumes of the input) of 25% solvent B (i.e., 75% solvent A; final 0.25 M KCl), collect the eluate (1.5 ml/fraction; total 20 fractions).
Change the valve position from “inject” to “load”.
Linear gradient flow (0.5 ml/min) ranging from 25% to 80% of solvent B with 15 ml, collect the eluate (0.5 ml/fraction; total 30 fractions).
Make small aliquot(s) of each fractionated sample on the UNO-Q column chromatography, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10).
Gradient fractionation on a hydrophobic column chromatography
Prepare the FPLC system and pre-packed hydrophobic column, SOURCE 15PHE PE 4.6/100, routinely
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGN1M as solvent B to inlet B, respectively.
Wash the column with 100% solvent A, and equilibrate with 100% solvent B.
Thaw the IREF-2-active fractions of the gradient-fractionated sample by the UNO-Q column chromatography and combine them as an input sample (= total 3 ml), and mix an equal volume of ice-cold buffer HGN2M, gradually. Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load the diluted supernatant (~6 ml) into the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the SOURCE 15PHE column (1.7 ml column volume) by reducing the concentration of (NH4)2SO4, as follows:
Isocratic flow (0.3 ml/min) with 15 ml (~2.5 volumes of the input) of 100% solvent B [i.e., 0% solvent A, final 1 M (NH4)2SO4], collect the eluate (0.75 ml/fraction; total 20 fractions).
Change the valve position from “inject” to “load”.
Linear gradient flow (0.3 ml/min) ranging from 100% to 0% of solvent B (i.e., from 0% to 100% of solvent A) with 12 ml, collect the eluate (0.75 ml/fraction; total 16 fractions).
Dialyze each fraction against 50 volumes of buffer HG for 5 h at 4 °C.
Make small aliquot(s) of each fractionated and dialyzed sample by the hydrophobic column chromatography, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10).
Gradient fractionation on a strong cation-exchanger column chromatography
Set up the FPLC system and pre-packed UNO S1 column, routinely.
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
Wash the column with 100% solvent B, and equilibrate with 3% solvent B (i.e., 97% solvent A).
Thaw the IREF-2-active fraction of the hydrophobic column chromatography, (i.e., unbound “flow-through” fraction). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load the supernatant (approximately 5 ml) to the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the UNO-S column (1.3 ml column volume) as follows:
Isocratic flow (0.4 ml/min) with 10 ml (2 volumes of the input) of 3% solvent B (i.e., 97% solvent A; final 0.03 M KCl), collect the eluate (0.65 ml/fraction; total 16 fractions).
Change the valve position from “inject” to “load”.
Linear gradient flow (0.5 ml/min) ranging from 3% to 100% of solvent B with 15.6 ml (12 column volumes), collect the eluate (0.65 ml/fraction; total 24 fractions).
Make small aliquot(s) of each fractionated sample on the UNO-S column chromatography, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10).
Gradient fractionation on a strong anion-exchanger column
Set up the FPLC system and pre-packed Mono-Q column, routinely.
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
Wash the column with 100% solvent B, and equilibrate with 35% solvent B (i.e., 65% solvent A).
Thaw the IREF-2-active fraction from the fractionated samples by the cation exchanger (UNO-S) column chromatography (i.e., unbound “flow-through” fraction). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load the supernatant (approximately 5 ml) to the sample loop and change the valve position to “inject”. Run the program for gradient fractionation on the Mono-Q column (1 ml column volume) as follows:
Isocratic flow (0.5 ml/min) with 10 ml (2 volumes of the input) of 35% solvent B (i.e., 65% solvent A; final 0.35 M KCl), collect the eluate (1 ml/fraction; total 10 fractions).
Change the valve position from “inject” to “load”.
Linear gradient flow (0.4 ml/min) ranging from 35% to 70% with 12 ml, collect the eluate (0.4 ml/fraction; total 30 fractions).
Make small aliquot(s) of each fraction from the Mono-Q column chromatography, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10 and Figure 4).
Figure 4. Profile of the fractions from Mono-Q column chromatography for IREF-2. Each Mono-Q fraction (fraction numbers 1-11) or input material for the Mono-Q column chromatography (i.e., unbound fraction of the UNO-S column chromatography at procedure C, 5) was individually added to the cell-free viral RNA synthesis reaction (upper panel). Each fraction was also subjected to 11.5% SDS-PAGE, and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. By comparing carefully between the IREF-2 activity level and the elution pattern of each peptide, two peptides (arrowheads A and B) migrated at 31 and 30 kDa are expected to be candidates for the IREF-2 activity. The molecular weight (kDa) positions are denoted on the left side of the panel. MWM: molecular weight marker.
Fractionation on a gel filtration column (see Note 11)
Set up the SMART system and gel filtration column, routinely.
Set degassed buffer HG as solvent A to inlet A and degassed buffer HGK as solvent B to inlet B, respectively.
Wash the column with 100% solvent B, and equilibrate with 20% solvent B (i.e., 80% solvent A).
Thaw the IREF-2-active fraction number 8 of the anion exchanger column chromatography (see Figure 4). Centrifuge at 20,000 x g for 5 min at 4 °C to remove insoluble material.
Load a small part of the supernatant (approximately 40 μl) into the sample loop and change the valve position to “inject”. Run the program for fractionation on the Superdex 75 gel filtration column (2.4 ml column volume) as follows:
Isocratic flow (50 μl/min) with 2.4 ml (1 column volume) of 20% solvent B (i.e., 80% solvent A; final 0.2 M KCl).
At 0.78 ml of the total flow, start collecting the eluate from the gel filtration column (30 μl/fraction).
Make small aliquot(s) of each fraction on the gel filtration column chromatography, and freeze in liquid N2 and store at -80 °C.
Check the IREF-2 activity present in each fraction (see Note 10 and Figure 5).
Figure 5. Profile of the fractions from gel filtration chromatography for IREF-2. Each gel-filtrated fraction (fraction numbers 1-23) or input material for the gel-filtration chromatography (i.e., the fraction number 8 of the Mono-Q column chromatography in step C6) was individually added to the cell-free viral RNA synthesis reaction (upper panel). Each fraction was also subjected to 11.5% SDS-PAGE, and polypeptides were visualized by silver staining (lower panel). The arrow indicates viral RNA replication products. The arrowheads indicate two candidate peptides responsible for the IREF-2 activity. The molecular weight (kDa) positions are denoted on the left side of the panel. MWM: molecular weight marker.
Identification of two polypeptides responsible for IREF-2 activity by mass spectrometry
Subject two Mono-Q fractions involving two candidate peptides, the fraction number 6 enriched with the peptide A and the fraction number 9 enriched with the peptide B (see Figure 4), to 11.5% SDS-PAGE.
Visualize the peptides separated in the gel by using a glutaraldehyde-free silver staining kit (for MS analysis).
Excise the gel pieces containing the bands of the peptide A and B, respectively (by using a clean razor blade).
Tryptic digestion in the gel pieces, followed by mass analysis (MALDI-TOF MASS).
Data analysis
Finally, pp32 (peptide A) and APRIL (peptide B) were identified as peptides derived from IREF-2 by database search using MS-Fit program in Protein Prospector (University of California San Francisco; http://prospector.ucsf.edu/prospector/mshome.htm). Top 3 hits of each mass analysis were summarized in Table 1. Observed mass (m/z) and theoretical mass for each digested peptide were summarized as “supplementary file 1 (http://elifesciences.org/content/4/e08939v2/supp-material1)” in the original article (Sugiyama et al., 2015).
Table 1. Top 3 hit proteins identified by MALDI-TOF MASS analyses
Notes
Several materials/reagents and equipment listed here, which were actually used in this protocol, are unavailable now. Of course, you can substitute these with other company’s or industry’s products.
For 8-L scale culture, it usually takes ~2 weeks from the beginning.
If more NEs are required for your purpose, a part of the cell culture (e.g., ~200 ml) should be aseptically kept and used for another round of large-scale culture.
All procedures for preparation of the nuclear extracts should be carried out as rapidly as possible at 4 °C or on ice.
Packed cell volume obtained from 8-L scale culture is usually 10-15 ml.
Packed nuclear volume obtained from 8-L scale culture is usually 8-12 ml.
The protein concentration usually results in about 5-10 mg/ml.
It is preferable to proceed continuously to the next procedure (i.e., first fractionation on the phosphocellulose column chromatography in this protocol) without freezing and thawing of NEs. To this end, the phosphocellulose column and FPLC system should be prepared on ahead to be ready-to-use (steps C1a and C1b).
Procedures of column chromatography (e.g., choice of column and fractionation way in stepwise or gradient, etc.) must be highly dependent on the chromatographic behavior and the purity of the protein of your interest at each column chromatography step. In addition to the column chromatography, you can utilize “ammonium sulfate precipitation” or “(glycerol or sucrose) density gradient centrifugation” method. For other cases of purification procedures, refer also to previous reports from our laboratory (Momose et al., 2001; Okuwaki et al., 2001; Momose et al., 2002; Kawaguchi and Nagata, 2007) and other laboratories.
Cell-free influenza virus RNA replication assay is carried out, as described briefly below: Single reaction (final volume of 20 μl) contains 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl2, 50 mM KCl, 1 mM DTT, 500 μM each ATP, UTP and CTP and 25 μM GTP, 5 mCi of [α-32P] GTP (3,000 Ci/mmol), 8 U of RNase inhibitor from human placenta, 10 ng of 53 nt-long influenza virus artificial model RNA template (termed “cRNA”) and 58 fmol of the viral RNA-dependent RNA polymerase prepared from influenza virion particles (termed “mnRNP”) as an enzyme source. In addition, NE fractions at each column chromatography step were individually added in the reactions to address whether IREF-2 activity was involved in the fraction, or not. After incubation at 30 °C for 2 h, reactions were terminated by extraction with phenol/chloroform followed by precipitation of RNA products with ethanol. The precipitated materials were subjected to 10% polyacrylamide gel electrophoresis in the presence of 7 M urea (Urea-PAGE), and visualized by autoradiography. For the detail of the cell-free viral RNA replication assay, see Sugiyama et al. (2015).
This gel filtration chromatography was performed for further confirmation that the peptides A and B (see Figure 4) were really responsible for the IREF-2 activity, not for further purification. Therefore, a small part of the IREF-2 active fraction (fraction number 8 in Figure 4) was subjected to the gel filtration chromatography with the Micro-Purification SMART system.
Recipes
Note: All buffers listed below should be prepared before use (not prepared long before) as ultra-pure “nuclease-free” grade.
Buffer A (used in steps B3 and B5)
10 mM HEPES-NaOH (pH 7.9 at 4 °C)
10 mM KCl
1.5 mM MgCl2
0.5 mM DTT (add 1 M stock just before use)
Buffer C (used in step B10)
20 mM HEPES-NaOH (pH 7.9 at 4 °C)
420 mM NaCl
1.5 mM MgCl2
200 mM EDTA
25% (v/v) glycerol
0.5 mM PMSF [add 1 M stock (in DMSO) just before use]
0.5 mM DTT (add 1 M stock just before use)
Buffer D (used in step B14)
20 mM HEPES-NaOH (pH 7.9 at 4 °C)
100 mM KCl
12.5 mM MgCl2
200 mM EDTA
25% (v/v) glycerol
0.5 mM PMSF [add 1 M stock (in DMSO) just before use]
0.5 mM DTT (add 1 M stock just before use)
Buffer HG (used in step C)
50 mM HEPES-NaOH (pH 7.9 at 4 °C)
20% (v/v) glycerol
Filtrate and degas with bottle top filter under vacuum.
Chill in 4 °C.
Add DTT (1 M stock, final 1 mM) just before use.
Buffer HGK (used in step C)
50 mM HEPES-NaOH (pH 7.9 at 4 °C)
1 M KCl
20% (v/v) glycerol
Filtrate and degas with bottle top filter under vacuum.
Chill in 4 °C.
Add DTT (1 M stock, final 1 mM) just before use.
Buffer HGN1M (used in step C4)
50 mM HEPES-NaOH (pH 7.9 at 4 °C)
1 M (NH4)2SO4
20% (v/v) glycerol
Filtrate and degas with bottle top filter under vacuum.
Chill in 4 °C.
Add DTT (1 M stock, final 1 mM) just before use.
Buffer HGN2M (used in step C4b)
50 mM HEPES-NaOH (pH 7.9 at 4 °C)
2 M (NH4)2SO4
20% (v/v) glycerol
1 mM DTT (add 1 M stock just before use)
Acknowledgments
The protocol for preparation of NEs preparation was based on Dignam et al. (1983). This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. N.).
References
Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5): 1475-1489.
Kawaguchi, A. and Nagata, K. (2007). De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM. EMBO J 26(21): 4566-4575.
Matsumoto, K., Nagata, K., Ui, M. and Hanaoka, F. (1993). Template activating factor I, a novel host factor required to stimulate the adenovirus core DNA replication. J Biol Chem 268(14): 10582-10587.
Momose, F., Basler, C. F., O'Neill, R. E., Iwamatsu, A., Palese, P. and Nagata, K. (2001). Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol 75(4): 1899-1908.
Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y. and Nagata, K. (2002). Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277(47): 45306-45314.
Nagata, K., Kawaguchi, A. and Naito, T. (2008). Host factors for replication and transcription of the influenza virus genome. Rev Med Virol 18(4): 247-260.
Okuwaki, M., Iwamatsu, A., Tsujimoto, M. and Nagata, K. (2001). Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins. J Mol Biol 311(1): 41-55.
Simon, R. H. and Felsenfeld, G. (1979). A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6(2): 689-696.
Schnitzler, G. R. (2001). Isolation of histones and nucleosome cores from mammalian cells. Curr Protoc Mol Biol Chapter 21: Unit 21 25.
Sugiyama, K., Kawaguchi, A., Okuwaki, M. and Nagata, K. (2015). pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. Elife 4: e08939.
Copyright: Sugiyama and Nagata. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Sugiyama, K. and Nagata, K. (2016). Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts. Bio-protocol 6(18): e1934. DOI: 10.21769/BioProtoc.1934.
Sugiyama, K., Kawaguchi, A., Okuwaki, M. and Nagata, K. (2015). pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. Elife 4: e08939.
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Microbiology > Microbe-host interactions > In vitro model
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Isolation and purification
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1,935 | https://bio-protocol.org/exchange/protocoldetail?id=1935&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots
TM Thomas J. Musielak
PB Patrick Bürgel
MK Martina Kolb
Martin Bayer
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1935 Views: 18043
Edited by: Samik Bhattacharya
Original Research Article:
The authors used this protocol in Dec 2015
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Original research article
The authors used this protocol in:
Dec 2015
Abstract
Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.
Keywords: Renaissance SR2200 Cell wall staining Arabidopsis thaliana Pollen tube Confocal microscopy
Materials and Reagents
Liquid blocker, “PAP pen” (Science Services, catalog number: N71310 )
Microscope slides (e.g., Superfrost; Carl Roth, catalog number: 1879.1 )
Double-sided adhesive tape (e.g., Reichelt Elektronik, Tesa Sande, catalog number: 05338 )
Syringe needle (e.g., Terumo, catalog number: NN-2425R ; VWR International, catalog number: HSWA4710005525 )
Microscope cover slips (e.g., Carl Roth, catalog number: H874.2 )
Pipette tips (200 μl)
Razor blade (e.g., VWR International, Darmstadt, catalog number: 233-5224 )
Fresh Arabidopsis plant material (growth conditions as described in Babu et al., 2013)
Deionized water
10% glycerol (e.g., Sigma-Aldrich, catalog number: 49782 )
1% (v/v) DMSO (e.g., Carl Roth, catalog number: 7029.2 )
0.05% (w/v) Triton-X 100 (e.g., Carl Roth, catalog number: 3051.2 )
3.75% (w/v) para-formaldehyde (e.g., Carl Roth, catalog number: 0335.2 )
PBS buffer (pH 7.4) (e.g., Carl Roth, catalog number: 9143.2 )
Optional: 4’,6-Diamidin-2-Phenylindol (DAPI) (e.g., Carl Roth, catalog number: 6335.1 )
SR2200 staining solution (see Recipes)
Note: SR2200 can be ordered from: Renaissance Chemicals Ltd, Unit 1 Blackwood Hall Business Park, North Duffield, Selby, UK. Contact: Howard Weaver, [email protected]
Equipment
Tweezers
Binocular (e.g., ZEISS, model: Stemi 2000 )
Adjustable pipette 20-200 μl (e.g., Gilson Pipetman-neo P200N; Carl Roth, catalog number: HY87.1 )
Vacuum pump (e.g., Vacuubrand PC2004 Vario)
Vacuum chamber (e.g., Kartell Labware 554)
Confocal laser scanning microscope with 405 nm laser and standard filter set for DAPI imaging (e.g., Leica TCS SP8) or spectral detector if double staining with DAPI is required (e.g., Zeiss LSM780NLO with 32 channel GaAsP array)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Musielak, T. J., Bürgel, P., Kolb, M. and Bayer, M. (2016). Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots. Bio-protocol 6(18): e1935. DOI: 10.21769/BioProtoc.1935.
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Category
Plant Science > Plant cell biology > Cell staining
Plant Science > Plant cell biology > Cell imaging
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1,936 | https://bio-protocol.org/exchange/protocoldetail?id=1936&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents
PD Patricia Dillenburg-Pilla
Carlos R. Zárate-Bladés
PS Phyllis B. Silver
RH Reiko Horai
Rachel R. Caspi
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1936 Views: 9720
Edited by: Ivan Zanoni
Reviewed by: Savita NairRon Saar-Dover
Original Research Article:
The authors used this protocol in Aug 2015
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The authors used this protocol in:
Aug 2015
Abstract
The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al., 2015). The protocol below describes the method to prepare intestinal protein-rich extracts that can be used in various in vitro and in vivo immunological studies.
Keywords: Microbiota Uveitis Autoimmune disease Antigenic mimicry Protein extract
Background
Intestinal microbiota represent a complex community of microbes that provide a wide variety of innate and adaptive stimulants. Their isolation and purification from stool samples has been performed and protocols have been published (Mueller and Pan, 2013; Verberkmoes et al., 2009; Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). Most of these protocols have been developed with the aim of performing proteomic studies for characterization of the microbiota. Consequently, although they emphasize protein yield and purity, they are time consuming and may include a protein denaturating step that affects protein structure (Verberkmoes et al., 2009) or use reagents (i.e., sodium azide, SDS, phenol) that are incompatible with subsequent cell culture based assays (Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). These characteristics are not desired when functional immunological assays are intended to be performed with the extracted proteins.
We have developed a simple and fast method that can be used to obtain protein-rich extracts from different areas of the intestine as well as from stool samples. The protocol does not include denaturating steps and the protein-rich extracts can be used in different in vitro and in vivo immunological assays with live cells, including T cell stimulation for proliferation and for adoptive transfer (see Data analysis section).
Materials and Reagents
50 ml centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352095 )
100 mm culture dish (Corning, Falcon®, catalog number: 353003 )
Sterile 12 ml syringe (COVIDIEN, MonojectTM, catalog number: 8881512878 )
23 G x 1” needle (COVIDIEN, MonojectTM, catalog number: 8881200383 )
3 ml sterile syringe (COVIDIEN, MonojectTM, catalog number: 8881513934 )
Sterile 1 ml syringe (COVIDIEN, MonojectTM, catalog number: catalog number: 8881501400 )
1.5 ml microtubes (Eppendorf, catalog number: 022-36-411-1 )
Syringe filter 0.22 μm, polyethersulfone, 33 mm, gamma sterilized (EMD Millipore, catalog number: SLGV033RS )
Sterile 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
PierceTM Coomassie plus Bradford protein assay kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236 )
10 mg/ml aprotinin (Sigma-Aldrich, catalog number: A1153 )
10 mg/ml leupeptin (Sigma-Aldrich, catalog number: L9783 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Protease inhibitor cocktail (see Recipes)
Equipment
CO2 mouse euthanasia chamber (with flow meter) (Euthanex, model: E-20028 )
-80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Revco UXF )
Analytical balance (Mettler-Toledo International, model: AE50 )
Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Legend XFR )
Refrigerated microcentrifuge (Eppendorf, model: 5424R )
Vortex-Genie 2
Sonicator (Heat systems, model: XL2010 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Dillenburg-Pilla, P., Zárate-Bladés, C. R., Silver, P. B., Horai, R. and Caspi, R. R. (2016). Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents. Bio-protocol 6(18): e1936. DOI: 10.21769/BioProtoc.1936.
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Category
Immunology > Mucosal immunology > Digestive tract
Immunology > Host defense > General
Biochemistry > Protein > Isolation and purification
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1,937 | https://bio-protocol.org/exchange/protocoldetail?id=1937&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling
Ryohei Iwata
Takuji Iwasato
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1937 Views: 9219
Edited by: Soyun Kim
Reviewed by: Shai BerlinEmmanuelle Berret
Original Research Article:
The authors used this protocol in Oct 2015
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Abstract
Dendritic spines are the post-synaptic structures that play a central role in excitatory synaptic transmission. Developmental spinogenesis relies on a variety of stimuli such as those derived from cell-cell communication and their downstream signaling. Here, we describe an in vitro assay of dendritic spine retraction using hippocampal slice culture, in which individual neurons are sparsely and brightly labeled by the Supernova method, for the study of molecular mechanisms of spine development.
Keywords: Spine Retraction assay EphA Spinogenesis
Materials and Reagents
Materials
Electrode (5 mm Φ platinum disk) (Nepa Gene, model: CUY650P5 )
Surgical needle (ELP, model: CR13-50 )
Glass capillary (Warner Instruments, model: GC150TF-10 )
NuncTM culture plate (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
Millicell culture plate inserts (EDM Millipore, catalog number: PICM03050 )
Confetti (LCR membrane filter) (EDM Millipore, catalog number: FHLC01300 )
Petri dish (60 mm) (Sigma-Aldrich, catalog number: Z721034 )
Note: This product has been discontinued.
Microcentrifuge tube (Sigma-Aldrich, catalog number: Z666505 )
Cover glass (MATSUNAMI GLASS)
Microscope glass slide (MATSUNAMI GLASS)
Aspirator tube assembly (Drummond Scientific, catalog number: 2-000-000 )
Sparse and bright labeling of hippocampal neurons using the in utero electroporation-based Supernova system (Mizuno et al., 2014)
Pregnant mouse (E13.5-E15.5)
Maxi-prep kit
Trypan blue solution (0.4%) (Sigma-Aldrich, catalog number: T8154 )
Supernova vector DNA solution (pK036.TRE-Flpe-WPRE-pA and pK037.CAG-FRT-STOP-FRT-TurboRFP-ires-tTA-WPRE-pA)
Somnopentyl (Pentobarbital sodium)
70% ethanol
Saline
Organotypic hippocampal slice culture
Mouse pup (P4-P5)
MEM with gluramax-1 (Thermo Fisher Scientific, catalog number: 41090-028 )
EBSS (Thermo Fisher Scientific, catalog number: 14155-048 )
D-glucose (NACALAI TESQUE, catalog number: 16805-35 )
Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
Nystatin (Thermo Fisher Scientific, GibcoTM, catalog number: 15340029 )
HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
Horse serum (Sigma-Aldrich, catalog number: H1270-500 ml )
Culture medium (See Recipes)
Slicing buffer(See Recipes)
In vitro retraction assay
EphrinA3-Fc (R&D Systems, catalog number: BT359 )
Human Fc fragment (Jackson ImmunoResearch, catalog number: 009-000-008 )
Goat anti-human IgG, Fc fragment specific (Jackson ImmunoReserach, catalog number: 109-001-008 )
Paraformaldehyde (NACALAI TESQUE, catalog number: 02890-45 )
PBS (-)
Vectashield mounting media with DAPI (Vector laboratories, catalog number: H-1200 )
Equipment
Sparse and bright labeling of hippocampal neurons using the in utero electroporation-based Supernova system.
Electroporator (Nepa Gene, model: Nepa21 )
Water bath
Puller (NARISHIGE Group, model: PC-10 )
Heating table (Leica Biosystems, model: HI1220 )
Surgical tools (scissor, 12 cm) (Fine Science Tools, catalog number: 14001-12 )
Surgical tools (scissor, 10.5 cm) (Fine Science Tools, catalog number: 14088-10 )
Surgical tools (forceps, 12 cm) (Fine Science Tools, catalog number: 11000-12 )
Surgical tools (forceps, 10 cm) (Fine Science Tools, catalog number: 11050-10 )
Surgical tools (ring forceps, 8.5 cm) (Fine Science Tools, catalog number: 11101-09 )
Organotypic hippocampal slice culture
LinearSlicer (Dosaka, model: PRO7 )
37 °C, 5% CO2 incubator
Pipette
In vitro retraction assay
25 °C incubator
Imaging and analysis
Confocal microscope (Leica Biosystems, model: TSC-SP5 )
Imaris filament tracer (Bitplane)
Software
Imaris
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Iwata, R. and Iwasato, T. (2016). In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling. Bio-protocol 6(18): e1937. DOI: 10.21769/BioProtoc.1937.
Iwata, R., Matsukawa, H., Yasuda, K., Mizuno, H., Itohara, S. and Iwasato, T. (2015). Developmental RacGAP α2-chimaerin signaling is a determinant of the morphological features of dendritic spines in adulthood. J Neurosci 35(40): 13728-13744.
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Category
Neuroscience > Cellular mechanisms
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1,938 | https://bio-protocol.org/exchange/protocoldetail?id=1938&type=0 | # Bio-Protocol Content
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Imaging Thick Lymph Node Tissue Sections
Jyh Liang Hor
Scott N. Mueller
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1938 Views: 12429
Edited by: Ivan Zanoni
Reviewed by: Francesca MingozziMeenal Sinha
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the spatial distribution of cellular events of interest (e.g., T cell clustering). This method can also readily be applied to other tissues, such as the spleen or skin.
Keywords: 2-photon microscopy Lymph nodes T lymphocytes Vibratome Immune response
Materials and Reagents
Razor blades, single edge, No.9 (0.22 mm) (VWR International, catalog number: 55411-050 )
Greiner CELLSTAR® 24-well flat-bottom plates (Sigma-Aldrich, catalog number: M8812 )
96-well round-bottom plates (Corning, catalog number: 3788 )
Durapore tape (3M, catalog number: 1538-1 )
SuperfrostTM Plus and ColorFrostTM glass slides (25 x 75 x 1.0 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4951PLUS4 )
Coverslips, No.1.5 (ProSciTech, catalog number: G425-2460 )
Agarose (Sigma-Aldrich, catalog number: A6013-250G )
Vetbond tissue adhesive (3M, catalog number: 1469SB )
Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
Protein block (Dako, catalog number: X0909 )
Normal donkey serum (NDS) (Jackson ImmunoResearch, catalog number: 017-000-121 )
Prolong gold antifade reagent (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36930 )
Antibodies [e.g., pacific blue anti-B220, RA3-6B2 (Biolegend, catalog number: 103230 ); goat anti-CD69 (R&D Systems, catalog number: AF2386 )]
Sodium periodate (NaIO4) (Sigma-Aldrich, catalog number: 311448 )
Sodium dihydrogen orthophosphate, monohydrate (NaH2PO4·H2O) (monobasic) (VWR, catalog number: 97062-412 )
Di-sodium hydrogen orthophosphate, anhydrous (Na2HPO4) (dibasic) (Ajax Finechem, catalog number: 621-500G )
L-lysine (Sigma-Aldrich, catalog number: L5501 )
Gelatin from porcine skin (Sigma-Aldrich, catalog number: G1890-100G )
Glycerol for molecular biology (≥ 99%) (Sigma-Aldrich, catalog number: G5516 )
16% paraformaldehyde aqueous solution (Electron Microscopy Sciences, catalog number: 30525-89-4 )
Periodate-lysine-paraformaldehyde (PLP) fixative (see Recipes)
P-buffer (see Recipes)
L-lysine (see Recipes)
5% gelatin/glycerol mix (see Recipes)
Equipment
Vibratome (Leica, model: VT1200S )
Dumont No.5 forceps (Fine Science Tools, catalog number: 11251-10 )
Scissors, straight, sharp (Roboz, catalog number: RS-6752 )
Soft bristled paint brush (e.g., size 2/0)
Microscope with tunable Coherent Chameleon Ti:Sa laser (ZEISS, model: LSM 710 NLO )
Note: This product has been discontinued (Replaceable item, e.g., ZEISS, model: LSM 800 ).
Scale for weighing 1 g
Microwave
Software
Imaris, Bitplane (http://www.bitplane.com/)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hor, J. L. and Mueller, S. N. (2016). Imaging Thick Lymph Node Tissue Sections. Bio-protocol 6(18): e1938. DOI: 10.21769/BioProtoc.1938.
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Category
Immunology > Immune cell imaging > Confocal microscopy
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1,939 | https://bio-protocol.org/exchange/protocoldetail?id=1939&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes
Jyh Liang Hor
Scott N. Mueller
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1939 Views: 8734
Edited by: Ivan Zanoni
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
Our protocol describes a simple method that allows tracking of dendritic cells (DC) migration from the flank skin to draining lymph nodes (LN) using a red fluorescent dye tetramethylrhodamine-5-isothiocyanate (TRITC). TRITC is a photostable dye that readily labels cells including DC in the skin and can survive repeated exposure of two-photon laser excitation for prolonged imaging duration. This method can be combined with various fluorescent antibody labels or transgenic mouse strains (such as CD11c-EYFP) to visualize distinct DC populations simultaneously.
Keywords: Dendritic cells Skin Lymph nodes Intravital imaging
Materials and Reagents
10-20 μl pipette tips
Paraffin film (Cole-Parmer, catalog number: PM996 )
Eppendorf tubes (Eppendorf, catalog number: 022364111 )
Felt-tip marker (Sharpie, fine point permanent marker)
Mouse (C57BL/6)
Tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC) (Thermo Fisher Scientific, catalog number: T490 )
Acetone (Chem Supply, catalog number: AL008-2.5L-P )
DMSO (Dimethyl sulfoxide) (Sigma-Aldrich, catalog number: D2650 )
Veet (sensitive skin hair removal cream) (Reckitt Benckiser)
Eye gel (e.g., Poly Visc, Alcon Laboratories)
Ilium Ketamil (Ketamine) (Troy Laboratories)
Ilium Xylazil-20 (Xylazine) (Troy Laboratories)
Ketamine/Xylazine solution (see Recipes)
Equipment
10-20 μl pipette
Spatula
Fine balance for weighing 0.1 μg
Wahl super trimmer (Wahl, catalog number: 5996415011081 )
Heat pad (Stoelting, model: 50300 )
Clippers
Tissue paper/gauze
Ruler
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Hor, J. L. and Mueller, S. N. (2016). Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes. Bio-protocol 6(18): e1939. DOI: 10.21769/BioProtoc.1939.
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Category
Immunology > Animal model > Mouse
Immunology > Immune cell function > Dendritic cell
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194 | https://bio-protocol.org/exchange/protocoldetail?id=194&type=1 | # Bio-Protocol Content
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Sucrose Gradient Analysis of Proteins
Hui Zhu
Published: Feb 5, 2012
DOI: 10.21769/BioProtoc.194 Views: 30194
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Abstract
The sedimentation rate of a protein in a linear sucrose gradient can be used to determine its S value when compared to proteins of known S values which are run in a parallel gradient. This can be used to estimate an approximate molecular weight of the protein and test the potential interaction of two proteins if they peak in the same gradient range.
Keywords: Sucrose gradient Molecular weight of the protein S value
Materials and Reagents
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Gelatin (Sigma-Aldrich, catalog number: G9136 )
Aldolase (Bio-Rad Laboratories)
Thyroglobulin (Bio-Rad Laboratories)
Sucrose solutions (see Recipes)
Equipment
Centrifuges (Beckman Falcon, TLS-55)
Gradient mixer (Sigma-Aldrich)
Stirring bar
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Biochemistry > Protein > Quantification
Cell Biology > Cell-based analysis > Transport
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1,940 | https://bio-protocol.org/exchange/protocoldetail?id=1940&type=0 | # Bio-Protocol Content
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Isolation of Intestinal Mesenchymal Cells from Adult Mice
Vasiliki Koliaraki
George Kollias
Published: Vol 6, Iss 18, Sep 20, 2016
DOI: 10.21769/BioProtoc.1940 Views: 23217
Edited by: Ivan Zanoni
Reviewed by: Meenal SinhaEmilie Besnard
Original Research Article:
The authors used this protocol in Dec 2015
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Dec 2015
Abstract
During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells from the adult mouse intestine.
Keywords: Mouse intestine Mesenchyme Fibroblasts Tissue digestion Collagenase
Materials and Reagents
Petri dishes (100 mm)
Note: Any Petri dish can be used, according to each researcher’s preference and availability.
Falcon tube
Transfer pipettes 3.5 ml (SARSTEDT, catalog number: 86.1172.001 )
50 ml conical tubes (SARSTEDT, catalog number: 62.547.004 )
Syringe filters, 0.2 μm pore size (SARSTEDT, catalog number: 83.1826.001 )
Cell strainers 70 μm (Corning, Falcon®, catalog number: 352350 )
Cell culture treated flasks (25 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136196 )
Cell culture treated flasks (175 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
Cell culture treated flasks (80 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178905 )
Mice
Ethanol
Note: Any ethanol can be used, according to each researcher’s preference and availability.
HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14170088 )
Antibiotic-antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
EDTA (Thermo Fisher Scientific, Fisher Scientific, catalog number: 327205000 )
DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: D9779 )
DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
Fetal bovine serum (FBS) (Biochrom, catalog number: S0115 )
Penicillin-streptomycin 100x (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-068 )
Phosphate-buffered saline (PBS) Dulbecco (10x) (Biochrom, catalog number: L1835 )
Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
Collagenase from Clostridium histolyticum, type XI (Sigma-Aldrich, catalog number: C7657 )
Dispase II, neutral protease grade II, from Bacillus polymyxa (Roche Diagnostics, catalog number: 04942078001 )
Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
FACS staining antibodies and material
Vimentin-A647 (Abcam, catalog number: ab194719 )
CD45-PE-Cy5 (BD, catalog number: 553082 )
Intracellular fixation & permeabilization buffer set (eBioscience, catalog number: 88-8824-00 )
HBSS/antibiotics (see Recipes)
HBSS/EDTA/DTT (see Recipes)
Digestion solution (see Recipes)
DMEM medium (see Recipes)
Equipment
Dissection kit
Two scissors (at least one medium size and one small)
Two forceps
Note: Any scissors and forceps can be used, according to each researcher’s preference and availability.
A 10 ml syringe equipped with a 21-gauge needle for flushing the intestine (BD, PlastipakTM, catalog number: 308063 )
Cell culture hood
Cell culture incubator, 37 °C, 5% CO2
Shaking water-bath
Centrifuge compatible with 50 ml conical tubes
Bright-field inverted microscope for cell culture
Multicolor FACS analyser (BD, model: BD FACS Canto II )
Software
FlowJo software (Tree Star Inc.)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Koliaraki, V. and Kollias, G. (2016). Isolation of Intestinal Mesenchymal Cells from Adult Mice. Bio-protocol 6(18): e1940. DOI: 10.21769/BioProtoc.1940.
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Category
Immunology > Immune cell isolation > Maintenance and differentiation
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1,941 | https://bio-protocol.org/exchange/protocoldetail?id=1941&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish
Alberto Rissone
Fabio Candotti
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1941 Views: 25447
Edited by: Jia Li
Reviewed by: Steven Tran
Original Research Article:
The authors used this protocol in Jul 2015
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Abstract
The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.
Keywords: Zebrafish Reactive Oxygen Species Oxidative stress
Materials and Reagents
10 cm sterile Petri dishes (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875711Z )
1.5 ml tubes (Eppendorf, catalog number: 022363204 )
35 mm TC-Treated culture dish (Corning, catalog number: 430165 )
Falcon round-bottom polystyrene 5 ml tubes (Corning, Falcon®, catalog number: 352003 )
Sterile 40 μm cell strainer (Corning, catalog number: 431750 )
35 mm glass bottom dish (Cellvis, catalog number: D35-20-1-N )
3 ml transfer pipette (Sigma-Aldrich, catalog number: Z135003 )
Zebrafish adults (wild-type or mutant strains)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138 )
ddH2O (Sigma-Aldrich, catalog number: W4502 )
DMEM/F-12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11330-032 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-036 )
Tricaine (Sigma-Aldrich, catalog number: A5040 )
10x trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15400-054 )
Tris-EDTA buffer solution (pH 9.0) (Sigma-Aldrich, catalog number: SRE0063 )
Collagenase P (Sigma-Aldrich, catalog number: COLLP-RO )
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
Hank's balanced salt solution (HBSS), with calcium and magnesium, no phenol red (HBSS/Ca/Mg) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025-092 )
MitoSOXTM red mitochondrial superoxide indicator (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M36008 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
7-aminoactinomycin D (7-AAD) (BD, BD PharmingenTM, catalog number: 559925 )
Annexin V-APC (BD, BD PharmingenTM, catalog number: 550474 )
Annexin V binding buffer, 10x concentrate (BD, BD PharmingenTM, catalog number: 556454 )
CellROX® green reagents, for oxidative stress detection (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10444 )
CellROX® green flow cytometry assay kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10492 )
CertifiedTM low-melting agarose (Bio-Rad Laboratories, catalog number: 1613111 )
Instant Ocean aquarium sea salt mixture (Spectrum Brand, Instant Ocean, catalog number: SS15-10 )
24x tricaine solution (see Recipes)
DMEM/F-12 medium (see Recipes)
60x E3 embryo medium stock solution (see Recipes)
Homogenization medium (see Recipes)
5 mM MitoSOX stock solution (see Recipes)
Equipment
Dissecting forceps - microdissection (DR Instruments, catalog number: 18 )
Ultrafine single deer hair with handle (Ted Pella, catalog number: 119 )
Thermoblock (Sigma-Aldrich, catalog number: Z605271 )
Manual pipette PR-200 (Mettler-Toledo, Rainin, catalog number: 17008652 )
Standard tabletop centrifuges (Eppendorf, catalog number: 022620401 )
Dissecting microscope (Leica, model: Leica S8 APO )
FACS-Fluorescence-activated cell sorter [e.g., FACSAria II equipped with FACSDiva software using the blue 488-nm and red 640-nm lasers (BD)]
Confocal microscope [e.g., LSM 510 NLO Meta system mounted on an Axiovert 200M microscope (Carl Zeiss) with a Plan-Apochromat 10x/0.45 or a Plan-Apochromat 20x/0.75 objective lens]
Software
FACSDiva software
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Rissone, A. and Candotti, F. (2016). Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish. Bio-protocol 6(19): e1941. DOI: 10.21769/BioProtoc.1941.
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Category
Cell Biology > Cell staining > Reactive oxygen species
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1,942 | https://bio-protocol.org/exchange/protocoldetail?id=1942&type=0 | # Bio-Protocol Content
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Peer-reviewed
Isolation, Culture, and Staining of Single Myofibers
Yann Simon Gallot
SH Sajedah M. Hindi
AM Aman K. Mann
Ashok Kumar
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1942 Views: 14283
Edited by: Jia Li
Reviewed by: Irit Adini
Original Research Article:
The authors used this protocol in Jan 2016
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Abstract
Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation.
Keywords: Single myofiber cultures Satellite cells Muscle injury Pax7 MyoD
Background
Although skeletal muscle is a fully differentiated post-mitotic tissue, it maintains intrinsic capability to regenerate in response to both genetic and acquired forms of muscle fiber damage (Le Grand and Rudnicki, 2007). Muscle regeneration in adults is mediated by a population of stem cells known as satellite cells which reside between the basal lamina and sarcolemma of myofibers in a mitotically quiescent state (Le Grand and Rudnicki, 2007). In response to muscle trauma, satellite cells become activated and proliferate to produce myoblasts that fuse with pre-existing fibers and with one another to repair or produce new myofibers. A small portion of satellite cells does not differentiate but instead reenters quiescence to maintain the stem cell pool. Satellite cells of all mammalian species express paired-box (Pax) transcription factor Pax7 which is also used as a critical marker to determine the fate of satellite cells in association with other myogenic factors such as MyoD (Le Grand and Rudnicki, 2007; Kuang et al., 2006; Kuang and Rudnicki, 2008).
The in vivo process of muscle regeneration with respect to satellite cell activation, proliferation, and differentiation can be partly recapitulated through suspension culture of myofiber explants (Rosenblatt et al., 1995; Shefer and Yablonka-Reuveni, 2005). The process of myofiber isolation which involves digestion of muscle tissue with matrix-degrading enzymes (e.g., collagenases) and mechanical shearing causes minor trauma which leads to the activation of satellite cells on myofibers. Immediately upon isolation, each myofiber is associated with a fixed number (Pax7+/MyoD-) of satellite cells resting in quiescence. At around 24 h in culture, satellite cells undergo their first round of cell division, through upregulating MyoD (Pax7+/MyoD+) and proliferate to form cell aggregates by 72 h. Satellite cells on cultured myofiber explants can either terminally differentiate (Pax7-/MyoD+) or self-renew (Pax7+/MyoD-) and return to quiescence (Le Grand and Rudnicki, 2007). The myofiber culture system has served as an excellent platform to study the fundamental properties of muscle stem cells and the effects of various genetic manipulations on the basic properties of satellite cells. One of the major advantages of isolated single myofiber cultures is that the physical interaction between the myofiber and satellite cells is preserved in the sense that satellite cells are still maintained beneath the basal lamina (Bischoff, 1986). Furthermore, suspension cultures of myofibers are routinely used to study the effects of various pharmacological compounds and overexpression or knockdown of specific proteins on self-renewal, proliferation, or differentiation of satellite cells (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Keire et al., 2013). They also provide a useful tool to study the motility of satellite cells on myofibers through live imaging (Siegel et al., 2009). In addition, a few investigators have used isolated single myofibers explants to prepare pure myoblast cultures in which the cells are mostly derived from the myofiber-associated satellite cells.
The isolation of single myofibers from flexor digitorum brevis (FDB) was first described by Bischoff (1986). This protocol was modified later by Rosenblatt et al. to allow handling of single myofibers after collagenase digestion (Rosenblatt et al., 1995). Since then several modifications have been proposed which improved the yield and the handling of the isolated myofibers (Shefer and Yablonka-Reuveni, 2005; Anderson et al., 2012; Verma and Asakura, 2011; Pasut, 2013). However, most of the published protocols still do not produce a sufficient number of myofibers in a single preparation primarily because of under or over digestion with collagenase and the loss of myofibers during picking, sub-culturing, or staining. The amount of time to digest a muscle from rodents may vary with genetic background and healthy versus diseased muscle. In our laboratory, we are now using a six-well tissue culture plate in which the digestion and physical separation of individual myofibers can be visually monitored under a phase contrast microscope. Furthermore, we have developed an improved protocol to stain the myofiber-associated satellite cells for various markers (Hindi et al., 2012; Hindi and Kumar, 2016; Ogura et al., 2013). In this article, we provide the detailed protocol for the isolation, culturing, and staining of myofiber-associated satellite cells for Pax7 and MyoD protein. The same protocol can be adapted for staining of other proteins in satellite cells of myofiber explant.
Materials and Reagents
Disposable borosilicate glass Pasteur pipets (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-678-20B )
Sterilization pouches (Thermo Fisher Scientific, Fisher Scientific, catalog number: 01-812-51 )
6-well plates (Corning, Falcon®, catalog number: 353046 )
0.22 µm filter (EMD Millipore, catalog number: SLGP033RS )
0.45 µm filter (EMD Millipore, catalog number: SLHV033RS )
24-well plates (Corning, Falcon®, catalog number: 353047 )
Adult mice (Mus musculus; ≥ 6-week old)
Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050-088 )
Dulbecco’s modified Eagle’s medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 )
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
Collagenase II (Worthington Biochemical Corporation, catalog number: LS004176 )
Ultra-pureTM water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-015 )
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
2, 2, 2-tribromoethanol (Avertin) (Sigma-Aldrich, catalog number: T48402 )
100% ethanol (Thermo Fisher Scientific, Fisher Scientific, catalog number: 2701 )
Tris base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
Recombinant human fibroblast growth factor-basic (bFGF) (PeproTech, catalog number: 100-18B )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
Chicken embryo extract (CEE) (Antibody Production Services, catalog number: MD-004E )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
100% Triton X-100 (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP151-500 )
Glycine (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP381-5 )
5% (w/v) sodium azide (Thermo Fisher Scientific, Fisher Scientific, catalog number: 71448-16 )
Primary antibody anti-MyoD (rabbit) (Santa Cruz Biotechnology, catalog number: sc-304 )
Primary antibody anti-Pax7 (mouse) (Developmental Studies Hybridoma Bank, catalog number: PAX7 )
Secondary antibody goat anti-rabbit Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, catalog number: A-11034 )
Secondary antibody goat anti-mouse Alexa Fluor® 568 conjugate (Thermo Fisher Scientific, catalog number: A-11004 )
4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D8417 )
Collagenase II (see Recipes)
Digestion medium (see Recipes)
Washing solution (see Recipes)
70% ethanol (see Recipes)
Basic fibroblast growth factor (bFGF) (see Recipes)
Chicken embryo extract (CEE) (see Recipes)
Myofiber growth medium (MfGM) (see Recipes)
4% paraformaldehyde (PFA) (see Recipes)
10% Triton X-100 (see Recipes)
Quenching solution (see Recipes)
Blocking solution (see Recipes)
Equipment
Water bath (Thermo Fisher Scientific, Fisher Scientific, catalog number: 15-462-15Q )
Microscope (Nikon Instruments, model: TE2000 )
CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3578 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Gallot, Y. S., Hindi, S. M., Mann, A. K. and Kumar, A. (2016). Isolation, Culture, and Staining of Single Myofibers. Bio-protocol 6(19): e1942. DOI: 10.21769/BioProtoc.1942.
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Category
Developmental Biology > Cell growth and fate > Myofiber
Stem Cell > Adult stem cell > Muscle stem cell
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1,943 | https://bio-protocol.org/exchange/protocoldetail?id=1943&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens
CG Charlotte Gruau
Patricia Trotel-Aziz
BV Bas Verhagen
SV Sandra Villaume
Fanja Rabenoelina
BC Barbara Courteaux
Christophe Clément
Fabienne Baillieul
Aziz Aziz
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1943 Views: 11733
Edited by: Arsalan Daudi
Reviewed by: Rupesh Paudyal
Original Research Article:
The authors used this protocol in Oct 2015
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Abstract
Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).
Keywords: Biocontrol Pseudomonas Grapevine Priming Botrytis
Materials and Reagents
25 mm culture tubes (VWR, catalog number: 212-0304 )
Conical tubes (50 ml) (Corning, Falcon®, catalog number: 352070 )
Sterile spatula (VWR, catalog number: 612-1561 )
Falcon® cell strainer 100 µm nylon (Corning, Falcon®, catalog number: 352360 )
1.5 ml microtubes (Treff, catalog number: 96.07246.9.01 )
2 ml microtubes (Treff, catalog number: 96.09329.9.01 )
Whatman paper ashless discs 90 mm diameter (Sigma-Aldrich, catalog number: WHA1441090 )
Petri dishes (90 x 14 mm) (VWR, catalog number: 391-0439 )
Grapevine (Vitis vinifera cv. Chardonnay 7535) plants obtained by micropropagation from nodal explants on agar Murashige & Skoog (MS) medium (Aziz et al., 2006) in 25 mm culture tubes
Pseudomonas fluorescens PTA-CT2 (Trotel-Aziz et al., 2008)
Botrytis cinerea conidia (Bc 630) isolated from infected grape at ripening by INRA Versailles, France
Distilled water from Aquadem blue range (Véolia Water Aquadem)
Distilled water sterilized with autoclave Avor Getinge
Ethanol (from Alcool absolu) (Charbonneaux Brabant, catalog number: 3077319001001 )
Murashige & Skoog medium (MS) (Duchefa Biochimie, catalog number: M0231.0001 )
Glycerol (VWR, catalog number: 24387-292 )
Luria Bertani liquid medium (LB) (Duchefa Biochimie, catalog number: L1703.0500 )
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
BactoTM agar (BD, catalog number: 214010 )
Agar tomato medium (see Recipes)
Equipment
Plant growth chamber with photoperiod (SANYO, model: MLR-351 H )
Horizontal laminar flow cabinet (ASTEC Microflow, model: Horizontal laminar flow workstation )
Long sterile metal clamp (VWR, catalog number: 232-0084 )
Magenta boxes (Sigma-Aldrich, catalog number: V8505 )
Microbiological safety cabinet (Angelantoni Life Science, model: VBH Compact )
Sterile pipettes (VWR, catalog number: VHPA23004 )
28 °C incubator shaker (New Brunswick Scientific, model: C24 )
Variable speed refrigerated centrifuge with rotor having number of rotor cavities x nominal largest load (capacity: 8 x 50 ml) (Eppendorf, model: 5804R )
Spectrophotometer (Bio-Rad Laboratories, model: SmartSpec 3000 )
Vortex mixer (Vortex Genie 2 untimed mixer) (VWR, catalog number: 58815-232 )
Malassez counting chamber (Marienfeld, catalog number: 0640610 )
Glass atomizer (VWR, catalog number: 552-0031 )
Camera (Nikon, model: D7000 )
Software
APS Assess 2.0 software (APS, Item No: 43696M5, ISBN 978-0-89054-369-6)
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant immunity > Disease bioassay
Plant Science > Plant immunity > Perception and signaling
Microbiology > Microbe-host interactions > In vivo model
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1,944 | https://bio-protocol.org/exchange/protocoldetail?id=1944&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
ASC-particle-induced Peritonitis
Lucia de Almeida
Andrea Dorfleutner
Christian Stehlik
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1944 Views: 6267
Edited by: Ivan Zanoni
Reviewed by: Achille BroggiMarco Di Gioia
Original Research Article:
The authors used this protocol in Aug 2015
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Abstract
In response to pathogen infection and tissue damage, inflammasome sensors such as NLRP3 and AIM2 are activated, which triggers PYRIN domain (PYD)-mediated ASC nucleation, followed by self-perpetuating ASC polymerization, which ultimately culminates in caspase-1 activation, interleukin (IL)-1β and IL-18 processing and release and pyroptosis (Ratsimandresy et al., 2013; Cai et al., 2014). Inflammasomes release not only cytokines, but also the polymeric ASC danger particles (pASC) by pyroptosis, which perpetuate and propagate inflammasome responses to bystander cells to engage cell intrinsic ASC and caspase-1 (Baroja-Mazo et al., 2014; Franklin et al., 2014). In this protocol we describe intraperitoneal injection of polymeric ASC particles as a danger signal and measure neutrophil infiltration and levels of the pro-inflammatory cytokine IL-1β by ELISA in the peritoneal lavage (de Almeida et al., 2015).
Keywords: Inflammasome Danger signal Inflammation Peritonitis NLRP3
Materials and Reagents
5 ml syringes (BD, catalog number: 309646 )
25 G x1 ½ needles (BD, catalog number: 305127 )
Conical tubes (15 ml) (Corning, Falcon®, catalog number: 352095 )
C57BL/6 mice, typically of 8-12 weeks old (male or female)
1 x 105 pASC-GFP particles generated from stable or transiently expressing HEK293 cells and sorted by flow cytometry as described (Fernandes-Alnemri et al., 2007; Fernandes-Alnemri and Alnemri, 2008)
1x Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV )
HEPES
KOH
Magnesium chloride (MgCl2)
EGTA
CHAPS
cOmplete protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001)
IL-1β ELISA kit (BD, catalog number: 559603 )
Lysis buffer (see Recipes)
Equipment
Centrifuge
Fluorescence microscope
Flow cytometry
Surgical instruments such as tweezers and scissors
Biosafety cabinet
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：de Almeida, L., Dorfleutner, A. and Stehlik, C. (2016). ASC-particle-induced Peritonitis. Bio-protocol 6(19): e1944. DOI: 10.21769/BioProtoc.1944.
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Category
Immunology > Animal model > Mouse
Cell Biology > Cell signaling > Stress response
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1,945 | https://bio-protocol.org/exchange/protocoldetail?id=1945&type=0 | # Bio-Protocol Content
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Peer-reviewed
In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis
Lucia de Almeida
Andrea Dorfleutner
Christian Stehlik
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1945 Views: 9905
Edited by: Ivan Zanoni
Reviewed by: Achille BroggiMarco Di Gioia
Original Research Article:
The authors used this protocol in Aug 2015
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Aug 2015
Abstract
Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP) (Whitfield and Trent, 2014). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the complex consisting of the LPS binding protein (LBP), CD14, MD-2 and Toll-like receptor 4 (TLR4) and the non-canonical inflammasome-linked caspases-4, 5 and 11, which in turn activate the canonical NLRP3 inflammasome (Shi et al., 2014; Hagar et al., 2013; Kayagaki et al., 2013; Hoshino et al., 1999; Poltorak, 1998; Nagai et al., 2002; Park et al., 2009; Ratsimandresy et al., 2013). In particular, the cytokine interleukin (IL)-1β produced in response to inflammasome activation has a crucial role in neutrophil recruitment through promoting neutrophil adhesion and migration (McDonald et al., 2010).This protocol allows studying of inflammatory response induced by LPS that affect neutrophil infiltration by tracking myeloperoxidase (MPO) activity in vivo (de Almeida et al., 2015).
Keywords: Inflammation Peritonitis Sepsis in vivo imaging Myeloperoxidase
Materials and Reagents
Insulin syringes (Thermo Fisher Scientific, catalog number: 14-841-31 )
0.22 μm filters
C57BL/6 mice, typically of 8-12 weeks old mice (male or female)
LPS E.coli 0111:B4 (Sigma-Aldrich, catalog number: L2630-100MG )
Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV )
XenoLight RediJect inflammation probe (PerkinElmer, catalog number: 760535 )
Luminol sodium salt (Sigma-Aldrich, catalog number: A4685 )
Isofluorane (Henry Schein, IsothesiaTM, catalog number: 10014450 )
5 mg/ml LPS (see Recipes)
20 mg/ml luminol sodium salt stock solution (see Recipes)
Equipment
Anesthesia machine (VetEquip, model: 901808 ) or similar anesthesia equipment
Rechargeable trimmer (Braintree Scientific, catalog number: VLP-323 75 )
Scale (Kent Scientific, catalog number: SCL66110 )
Biosafety cabinet
IVIS spectrum (PerkinElmer, model: 124262 ) or a comparable luminescence imaging equipment
Software
Living Image software (PerkinElmer)
Procedure
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Category
Immunology > Animal model > Mouse
Cell Biology > Cell movement > Cell migration
Cell Biology > Cell signaling > Stress response
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1,946 | https://bio-protocol.org/exchange/protocoldetail?id=1946&type=0 | # Bio-Protocol Content
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Peer-reviewed
Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
FF Fabienne C. Fiesel
Roman Hudec
Wolfdieter Springer
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1946 Views: 7980
Edited by: Ralph Bottcher
Reviewed by: Qiangjun ZhouVanesa Olivares-Illana
Original Research Article:
The authors used this protocol in Sep 2015
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Abstract
This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.
Keywords: PINK1 Parkin Ubiquitin Mitophagy Parkinson's disease
Background
In cells, PINK1 is stabilized and activated by mitochondrial membrane depolarization and other forms of stress that lead to mitochondrial damage. Activated PINK1 phosphorylates ubiquitin, which acts as the receptor for the cytosolic E3 ubiquitin ligase Parkin on the mitochondrial surface. Phosphorylation of Parkin by PINK1 is required for full activity of Parkin towards mitochondrial substrates. The presence of active pS65-Parkin amplifies in a feed-forward mechanism the amount of pS65-ubiquitin on mitochondria, which acts as the mitophagy tag. Eventually, damaged mitochondria are being recognized by autophagy adapters and will be degraded by the proteasome and by autophagy (mitophagy). This crucial mitochondrial quality control pathway promotes turnover of mitochondria and prevents accumulation of dysfunctional mitochondria that can lead to cellular degeneration. Loss-of-function mutations in either PINK1 or Parkin are associated with early-onset Parkinson’s disease.
In cell-free ubiquitination assays, the presence of pS65-Ub activates the E3 ligase activity of Parkin also in the absence of active PINK1. While many groups use their own purified recombinant proteins for in vitro PINK1 kinase assays, this optimized protocol describes the in vitro phosphorylation of ubiquitin and Parkin with commercially available recombinant proteins.
Materials and Reagents
Recombinant proteins
Active PINK1 [MBP-tagged] from Tribolium castaneum (Ubiquigent, catalog number: 66-0043-050 )
Inactive PINK1 D359A [MBP-tagged] from Tribolium castaneum (Ubiquigent, catalog number: 66-0044-050 )
Parkin (untagged) (Ubiquigent, catalog number: 63-0048-025 )
Ubiquitin (untagged) (BOSTON BIOCHEM, catalog number: U-100H )
Ubiquitin N-terminal biotin (BOSTON BIOCHEM, catalog number: UB-560 )
Biotinylated poly-ubiquitin chains (K48-linked) (BOSTON BIOCHEM, catalog number: UCB-230 )
Poly-ubiquitin chains (K63-linked) (Biotinylated) (BOSTON BIOCHEM, catalog number: UCB-330 )
Chemicals for buffers
HEPES (Sigma-Aldrich, catalog number: H4034 )
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
EDTA (EMD Millipore, catalog number: EX0539 )
ATP (AppliChem, catalog number: A1348 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M0250 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60370 )
Tris (Santa Cruz Biotechnology, catalog number: sc-3715 )
Hydrochloric acid (37%) (EMD Millipore, catalog number: 1003172500 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP2291 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Bromophenol blue sodium salt (Sigma-Aldrich, catalog number: B5525 )
Glycine (Sigma-Aldrich, catalog number: G7126 )
Methanol (Pharmco-Aaper, catalog number: 339000000 )
Dry milk powder
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP358-10 )
Tween-20 (Sigma-Aldrich, catalog number: P1379 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
10x kinase buffer (see Recipes)
6x SDS loading buffer (see Recipes)
SDS running buffer (see Recipes)
Blotting buffer (see Recipes)
TBST (see Recipes)
Resolving gel buffer (see Recipes)
14% resolving phos-tag gel (recipe for mini gel) (see Recipes)
8% resolving phos-tag gel (recipe for mini gel) (see Recipes)
Stacking gel buffer (see Recipes)
Stacking gel (recipe for two mini gels) (see Recipes)
Phos-tag gel washing buffer 1 (see Recipes)
Phos-tag gel washing buffer 2 (see Recipes)
Phosphate buffer saline (PBS) (see Recipes)
Antigen retrieval solution (see Recipes)
SDS-PAGE/Western blot
8-16% Tris-glycine gels (for Parkin) (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC60485BOX )
16% Tris-glycine gels (for ubiquitin) (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC64985BOX )
Pre-stained protein ladder (Bio-Rad Laboratories, catalog number: 1610394 )
PVDF membrane (EMD Millipore, catalog number: IPVH00010 )
pS65-Parkin antibody (Abcam, catalog number: ab154995 )
pS65-Ubiquitin antibody (EMD Millipore, catalog number: ABS1513-I or BOSTON BIOCHEM, catalog number: A-110 )
Monoclonal Parkin antibody Prk8 (Cell Signaling Technology, catalog number: 4211 )
Monoclonal Ubiquitin antibody (LifeSensor, catalog number: VU-101 )
PierceTM streptavidin, HRP-linked (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21130 )
HRP-conjugated anti-rabbit antibodies (Jackson Immunoresearch, catalog number: 111-035-003 )
HRP-conjugated anti-mouse antibodies (Jackson Immunoresearch, catalog number: 115-035-003 )
Western blot detection reagent (EMD Millipore, catalog number: WBKLS0500 )
X-ray films (Genesee Scientific, catalog number: 30-101L )
Acrylamide:Bis-acrylamide 19:1, solution 40% (w/v) (EMD Millipore, catalog number: 1290-OP )
Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 96468 )
Ammonium persulfate [(NH4)2S2O8] (Sigma-Aldrich, catalog number: A9164 )
TEMED (Santa Cruz Biotechnology, catalog number: sc-29111 )
Phos-tagTM acrylamide AAL-107 (Wako Pure Chemical Industries, catalog number: 304-93521 )
25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G6257 )
Equipment
Standard bench-top centrifuge
Heated shaker for microtubes [e.g., Thermomixer (Eppendorf, catalog number: 5384000020 )]
SDS-PAGE gel electrophoresis and blotting boxes (e.g., VWR International, Peqlab, catalog number: 45-1010-C and Bio-Rad Laboratories, catalog number: 1704070 )
Orbital shaker for Western blots
X-ray film processor or camera detection system (e.g., GE Healthcare, catalog number: 28-9558-10 ) to visualize chemiluminescent Western blot signal
Software
ImageJ or ImageStudio Lite
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Fiesel, F. C., Hudec, R. and Springer, W. (2016). Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate. Bio-protocol 6(19): e1946. DOI: 10.21769/BioProtoc.1946.
Fiesel, F. C., Ando, M., Hudec, R., Hill, A. R., Castanedes-Casey, M., Caulfield, T. R., Moussaud-Lamodiere, E. L., Stankowski, J. N., Bauer, P. O., Lorenzo-Betancor, O., Ferrer, I., Arbelo, J. M., Siuda, J., Chen, L., Dawson, V. L., Dawson, T. M., Wszolek, Z. K., Ross, O. A., Dickson, D. W. and Springer, W. (2015). (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation. EMBO Rep 16(9): 1114-1130.
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Category
Neuroscience > Cellular mechanisms > Mitochondria
Biochemistry > Protein > Modification
Biochemistry > Protein > Activity
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1,947 | https://bio-protocol.org/exchange/protocoldetail?id=1947&type=0 | # Bio-Protocol Content
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Peer-reviewed
Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay
LW Laura Winters
NT Nithya Thambi
JA Julian Andreev
FK Frank Kuhnert
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1947 Views: 13817
Edited by: Lee-Hwa Tai
Reviewed by: Anita Umesh
Original Research Article:
The authors used this protocol in Oct 2015
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Abstract
Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. Here, we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints.
Background
Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a physiological process that occurs during wound healing and normal development. Angiogenesis is a complex and highly regulated process involving the tight coordination of endothelial cell proliferation, differentiation, migration, matrix adhesion, and cell-to-cell signaling. Angiogenesis is also critically involved in tumor development and metastasis. Indeed, targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated clinical benefit. Since tumors do eventually develop resistance to VEGF-targeted therapy, there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) (Nakatsu et al., 2007; Nakatsu and Hughes, 2008; Nehls and Drenckhahn, 1995) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. This assay recapitulates key aspects of angiogenesis such as lumen formation, endothelial cell polarization and dependency on stromal cells, and is correlative with the activities of angiogenesis inhibitors as observed in in vivo tumor studies (Figures 1 and 2) (Eichten et al., 2013; Holash et al., 2012; Kuhnert et al., 2015; Noguera-Troise et al., 2006). Here we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints, such as the number of endothelial cells or branch points, as well as sprout length and area (Figure 3). To illustrate the spectrum of treatment outcomes in the FBA, the effects of three different angiogenesis inhibitors [aflibercept, Dll4 blocking monoclonal antibody (Dll4 MAB) and anti-Integrin a6 antibody GOH3] on endothelial sprouting have been included in the protocol.
Materials and Reagents
50 ml conical tubes (Corning, Falcon®, catalog number: 352070 )
Cytodex-3 beads (GE Healthcare, Amersham Pharmacia Biotech, catalog number: 17-0485-01 )
Aspirator
FACS tubes (5 ml) (Corning, Falcon®, catalog number: 352063 )
15 ml conical tubes (Conring, Falcon®, catalog number: 352099 )
1.5 ml centrifuge tube
24 well plate (Corning, Falcon®, catalog number: 351147 )
0.22 μm filter (VWR, catalog number: 28145-501 )
Human umbilical vein endothelial cells (HUVEC) and HUVEC complete media (Lonza, catalog numbers: C2517A and CC3162 )
Note: Optimal at passage 2-4.
Normal human lung fibroblasts (NHLF) (Lonza, catalog number: CC2512 )
Sigmacote siliconizing reagent (Sigma-Aldrich, catalog number: SL2-25ML )
Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230-162 )
DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-141 )
Fibrinogen from bovine plasma (Sigma-Aldrich, catalog number: F8630-1G )
Thrombin from bovine plasma (Sigma-Aldrich, catalog number: T3399-1KU )
Note: This product has been discontinued.
Aprotinin (Sigma-Aldrich, catalog number: A1153-10MG )
Clonetics EGM-2 bullet kit (Lonza, catalog number: CC3162 )
Trypsin/EDTA (0.025% trypsin/0.75 mM EDTA) (EMD Millipore, catalog number: SM-2004-C )
Aflibercept (VEGF-Trap) (Regeneron Pharmaceuticals)
Dll4 blocking monoclonal antibody (Dll4 MAB) (Regeneron Pharmaceuticals)
Rat anti-human integrin α-6 antibody, GoH3 (BD, BD PharmingenTM, catalog number: 555734 )
Paraformaldehyde (PFA) (16%) (Electron Microscopy Sciences, catalog number: 15710 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787-100ML )
Phalloidin-Tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) (Sigma-Aldrich, catalog number: P1951-.1MG )
FGM-2 bullet kit (Lonza, catalog number: CC3132 )
Hoechst 33258, pentahydrate, bis-benzimide (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: H3569 )
Fibrinogen solution (see Recipes)
Thrombin stock solution (see Recipes)
Aprotinin stock solution (see Recipes)
Equipment
Siliconized glass bottles (Corning, PYREX®, catalog number: 1395-100 )
Laminar flow hood
Water bath (PolyScience, catalog number: WB05A11B )
T25 flasks (Corning, catalog number: 430639 )
Centrifuge
Incubator
P1000 pipette
24 well glass bottom sensoPlate (Greiner Bio One, catalog number: 662892 )
Cell counter (Nexcelom BioScience, model: Cellometer Auto 1000 )
Microscope (Nikon, model: Eclipse Ti-S )
ImageXpress® MICRO XL (Molecular Devices)
Software
MetaXpress (Molecular Devices)
Note: MetaXpress software (MX) from Molecular Devices is optimized to perform with the ImageXpressMICRO imaging systems. MX is used both to control image acquisition and to perform image analysis. Necessary features of MX utilized in FBA image acquisition and analysis are: 1) Image acquisition: Laser-based autofocus, Z-stack acquisition; 2) Image analysis: 'Tube Formation' image analysis application module, an interactive Custom Module.
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Winters, L., Thambi, N., Andreev, J. and Kuhnert, F. (2016). Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay. Bio-protocol 6(19): e1947. DOI: 10.21769/BioProtoc.1947.
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Category
Cancer Biology > Angiogenesis > Cancer therapy
Cell Biology > Cell structure > Cell surface
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1,948 | https://bio-protocol.org/exchange/protocoldetail?id=1948&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Cellular Copper in Rhodobacter capsulatus by Atomic Absorption Spectroscopy
Petru-Iulian Trasnea
Dorian Marckmann
Marcel Utz
Hans-Georg Koch
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1948 Views: 9521
Edited by: Valentine V Trotter
Reviewed by: Filipa Vaz
Original Research Article:
The authors used this protocol in Apr 2016
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Apr 2016
Abstract
Copper is an essential micronutrient and functions as a cofactor in many enzymes such as heme-Cu oxygen reductases, Cu-Zn superoxide dismutases, multi-copper oxidases and tyrosinases. However, due to its chemical reactivity, free copper is highly toxic (Rae et al., 1999) and all organisms use sophisticated machineries for controlling uptake, storage and export of Cu. The strict control of the cellular Cu homeostasis prevents toxic effects but sustains synthesis of cuproproteins. Monitoring the copper levels within the cell and within different cellular compartments is an essential approach for identifying the contribution of different proteins in maintaining the cellular copper equilibrium. Therefore, whole cells and whole-cell lysates, which can be further fractionated into cytoplasm and periplasm, were digested and the protein concentration was determined by Lowry assay. Subsequently, the copper content was measured by atomic absorption spectroscopy (AAS) and the Cu content per mg of protein was calculated. This provides a simple and cost-effective method of producing quantifiable results about the cellular Cu content. To exemplify this method, we used the phototrophic α-proteobacterium Rhodobacter capsulatus, which is commonly used as a model organism for studying Cu-trafficking in bacterial cells (Ekici et al., 2012).
Keywords: Copper homeostasis Cbb3-type cytochrome oxidase Rhodobacter capsulatus Copper chaperones Copper-dependent Enzymes
Background
Due to a growing interest in cellular Cu homeostasis different methods for measuring the cellular Cu content have been developed during the past years. They include electrochemical and fluorimetric protocols, inductively coupled plasma mass spectroscopy (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES), electron microprobe analyses (EMPA), X-ray absorption spectroscopy (XAS) or synchrotron-based X-ray fluorescent microscopy (SXRF) (reviewed in Ralle et al., 2009). Although these methods allow for a reliable and accurate determination of Cu in biological and environmental samples, they usually require advanced experimental set-ups and are not generally suited for analyzing a large number of samples. Atomic absorption spectroscopy (AAS) is a well established and widely available method that allows for a quick, sensitive and cost-effective Cu determination. It is suitable for determining Cu in whole cells but also in subcellular extracts.
Materials and Reagents
Petri dishes (60 x 15 mm) (SARSTEDT, catalog number: 82.1194.500 )
50 ml Falcon tubes (SARSTEDT, catalog number: 62.559.001 )
15 ml Falcon tubes (SARSTEDT, catalog number: 62.554.502 )
0.45 µm filters (Carl Roth, catalog number: P667.1 )
10 ml syringes (Carl Roth, catalog number: C542.1 )
Rhodobacter capsulatus wild type (MT1131) or mutant strains
BactoTM peptone (BD, catalog number: 211820 )
BactoTM yeast extract (BD, catalog number: 212720 )
Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239.1 )
Magnesium chloride hexahydrate (MgCl2·6H2O) (Carl Roth, catalog number: 2189.2 )
MPYE agar media (1.5% agar in MPYE medium; 25 ml/Petri dish)
Chelex® 100 resin (Bio-Rad Laboratories, catalog number: 1422832 )
Tris/HCl, pH 7.5 (Carl Roth, catalog number: 5429.2 ; P074.1 )
Sucrose (MP Biomedicals, catalog number: 04821713 )
Lysozyme (Sigma-Aldrich, catalog number: L2879 )
EDTA (Carl Roth, catalog number: 8043.2 )
Lowry protein assay reagents [Reagent A, Folin & Ciocalteu’s phenol reagent (Sigma-Aldrich, catalog number: F9252 ), 1% SDS/0.1 N NaOH]
SDS (SERVA Electrophoresis, catalog number: 20765.03 )
Protein assay bovine serum albumin (Carl Roth, catalog number: 8076.4 ), standards (0, 0.02, 0.04, 0.08, 0.12, 0.2 mg/ml protein)
Sodium carbonate (Na2CO3) (Carl Roth, catalog number: 8563.1 )
Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.1 )
Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Carl Roth, catalog number: P024.1 )
Na-tartrate (EMD Millipore, catalog number: 106663 )
53% nitric acid in ultra-pure water (Carl Roth, catalog number: 9274.1 )
30% hydrogen peroxide (Carl Roth, catalog number: 9681.1 )
Ultra-pure water (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTMGenPureTM )
Palladium (II) chloride (VWR, catalog number: AA11034-09 )
Magnesium chloride (MgCl2) (VWR, catalog number: AA42843-22 )
Copper standards [(20, 40, 60 ppb Cu in ultra-pure water; diluted from TraceCert copper standard for AAS (1,000 mg/ml Cu in 2% nitric acid, prepared with high purity Cu metal)] (Sigma-Aldrich, catalog number: 38996 )
MPYE media (see Recipes)
Cu-free MPYE (see Recipes)
Cu-free water (see Recipes)
Spheroplast buffer (see Recipes)
Lysis buffer (see Recipes)
Reagent A for Lowry assay (see Recipes)
AAS modifier solution (see Recipes)
Equipment
1.5 ml cuvettes (Carl Roth, catalog number: Y195.1 )
Magnetic stir plate (Heidolph, model: Hei-VAP )
Sterile inoculation loop (Carl Roth, catalog number: 6163.1 )
Shaking incubator multitron standard for bacterial growth at 35 °C (Infors, mode: INFORS HT )
250 ml Erlenmeyer flasks (Carl Roth, catalog number: K184.1 )
Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Lynx 6000 )
Microscope at 100x magnification with numerical aperture 1.4 (OLYMPUS, model: BX51 )
1,000 µl automatic pipet (Gilson, catalog number: F123602 )
Incubator for sample incubation at 60 °C and 80 °C (Kühner, model: ISF-1X )
Atomic absorption spectrophotometer (PerkinElmer, model: 4110 ZL Zeeman )
VIS spectrophotometer able to read OD660 and OD685 (e.g., GE Healthcare Life Science, model: Ultrospec 3100 Pro )
Software
Excel 2007 (Microsoft)
Perkin-Elmer AAWinLab software
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Trasnea, P., Marckmann, D., Utz, M. and Koch, H. (2016). Measurement of Cellular Copper in Rhodobacter capsulatus by Atomic Absorption Spectroscopy . Bio-protocol 6(19): e1948. DOI: 10.21769/BioProtoc.1948.
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Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Ion
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1,949 | https://bio-protocol.org/exchange/protocoldetail?id=1949&type=0 | # Bio-Protocol Content
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Peer-reviewed
Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts
Haydar Karakaya
KÖ Kübra Özkul
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1949 Views: 13205
Edited by: Maria Sinetova
Reviewed by: Dennis Nürnberg
Original Research Article:
The authors used this protocol in Nov 2015
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Nov 2015
Abstract
Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6-phosphoglucono-δ-lactone and transfers one electron to NADP+ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the production of NADPH. The increase in NADPH concentration results in an increase in absorbance at 340 nm. To assay G6PDH activity, therefore, production of NADPH is determined by measuring increase in absorbance at 340 nm spectrophotometrically. This increase rate is then converted to unit of activity and specific activity of G6PDH. In this procedure, a generalized method is given for bacterial G6PDH assays emphasizing on a cyanobacterium Synechocystis sp. PCC6803 (Schaeffer and Stanier, 1978; Karakaya et al., 2008, 2012) and a heterotrophic bacterium E.coli (Hylemon and Phibbs, 1972; Barnel et al., 1990).
Keywords: Cyanobacteria Glucose-6-phosphate dehydrogenase Specific activity Cell-free extract
Materials and Reagents
1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022363204 )
Micropipette tips (200 μl) (Sigma-Aldrich, catalog number: CLS4866 )
Micropipette tips (1,000 μl) (Sigma-Aldrich, catalog number: CLS4868 )
Glass beads (unwashed) (212-300 μm) (Sigma-Aldrich, catalog number: G9143 )
1.5 ml polystyrene spectrophotometer cuvettes with 10 mm path length (Sigma-Aldrich, catalog number: C5416 )
Parafilm (Sigma-Aldrich, catalog number: P7793 )
Bacterial cells
Note: Amount of the cell depends on how many assays will be carried out. Supernatant yielded from a cell pellet of 50 ml well-grown cyanobacterial culture (OD750 ≥ 1.0) and 10 ml overnight grown Escherichia coli will be sufficient for about 20 assays depending on what volume is used for each assay.
Trizma® base (Sigma-Aldrich, catalog number: T1503 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Glucose-6-phosphate disodium salt (G6P) (Sigma-Aldrich, catalog number: G7250 )
Potassium phosphate dibasic trihydrate (K2HPO4·3H2O) (Sigma-Aldrich, catalog number: P5504 )
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
Hydrochloric acid (HCl) (36.5-38.0%) (Sigma-Aldrich, catalog number: H1758 )
Maleic acid (Sigma-Aldrich, catalog number: M0375 )
β-nicotinamide adenine dinucleotide phosphate hydrate (NADP+) (Sigma-Aldrich, catalog number: N5755 )
Paraffin
50 mM Tris-maleate solution (see Recipes)
Extraction buffers
Tris-maleate buffer, pH 6.8 for Synechocystis sp. PCC6803 (see Recipes)
Potassium phosphate buffer, pH 6.8 for E.coli (see Recipes)
Assay buffers
a.Tris-maleate buffer, pH 7.4 for Synechocystis sp. PCC6803 (see Recipes)
b.Tris-HCl buffer, pH 8.0 for E.coli (see Recipes)
500 mM Tris-HCl stock solution (pH 8.0) (see Recipes)
500 mM G6P solution (see Recipes)
100 mM NADP+ solution (see Recipes)
Equipment
Micropipettes (20-200 μl capacity) (Nichiryo, catalog number: 00-NPX2-200 )
Micropipettes (100-1,000 μl capacity) (Nichiryo, catalog number: 00-NPX2-1000 )
Microcentrifuge, 1.5 ml Eppendorf tube rotor and at least 10,000 x g force (Sigma-Aldrich, Hettich®, model: MIKRO120 )
FastPrep FP120 (BioSurplus, Thermo-Savant, model: FP120 )
Light microscope
Vortex mixer (Bibby Scientific, Stuart, model: SA8 )
Balance (Precision Weighing Balances, Salter Brecknell, model: ESA-150 )
pH meter (Hanna Instruments, model: HI5221 )
Vis-Spectrophotometer with 1.5 ml cuvette holder (Shimadzu Scientific Instruments, model: UV-1800 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Karakaya, H. and Özkul, K. (2016). Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts. Bio-protocol 6(19): e1949. DOI: 10.21769/BioProtoc.1949.
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Category
Biochemistry > Protein > Activity
Microbiology > Microbial biochemistry > Protein
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195 | https://bio-protocol.org/exchange/protocoldetail?id=195&type=0 | # Bio-Protocol Content
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Peer-reviewed
Propidium Iodide Staining of Cells for FACS Analysis
Hui Zhu
Published: Vol 2, Iss 11, Jun 5, 2012
DOI: 10.21769/BioProtoc.195 Views: 56710
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Abstract
Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA staining can be used to study the cell cycle. Relative DNA content shows the proportion of cells in G1, G2 and S phases. Apoptotic cells show characteristic smear on DNA staining. Here a protocol to stain cells by PI is described.
Keywords: Propidium iodide FACS Cell cycle
Materials and Reagents
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
RNase A (Sigma-Aldrich, catalog number: R4642 )
Phosphate buffered saline (PBS)
70% EtOH
10 % Triton X-100 (see Recipes)
PI stock solution (see Recipes)
Equipment
Standard table-top centrifuges
FACS machine
Incubator
Procedure
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhu, H. (2012). Propidium Iodide Staining of Cells for FACS Analysis. Bio-protocol 2(11): e195. DOI: 10.21769/BioProtoc.195.
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Category
Cancer Biology > Proliferative signaling > Cell biology assays
Cancer Biology > General technique > Cell biology assays
Cell Biology > Cell staining > Nucleic acid
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1,950 | https://bio-protocol.org/exchange/protocoldetail?id=1950&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Measuring Rat Serum Osmolality by Freezing Point Osmometry
Jerneja Stare
CB Charles W. Bourque
Published: Vol 6, Iss 19, Oct 5, 2016
DOI: 10.21769/BioProtoc.1950 Views: 13429
Edited by: Soyun Kim
Reviewed by: Emmanuelle BerretAntoine de Morree
Original Research Article:
The authors used this protocol in Sep 2015
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Sep 2015
Abstract
Blood serum or plasma osmolality is the measure of electrolyte to water balance in the body’s circulation, and is tightly regulated in physiological states in order to maintain normal levels of serum solute (Bourque, 2008). Osmolality is defined as the number of osmoles of solute per kg of water (mOsm/kg) (Dufour, 1993) and can be measured using different techniques that rely on the colligative properties of the solution. The most commonly used in lab settings are vapour pressure and freezing point osmometry, which are relatively quick and easy to perform. Freezing point osmometry is preferred because it is insensitive to volatile compounds, such as alcohol, that may be present in the solution. Measurement of serum or plasma osmolality is clinically relevant for a number of conditions and diseases, including hypernatremia, diabetic ketoacidosis, and the syndrome of inappropriate antidiuresis (Ellison, 2013; Lupsa and Inzucchi, 2013; Reddi, 2013). In this protocol, we describe the measurement of serum osmolality in rats using the freezing point osmometry technique as originally outlined in our previous study of osmoregulatory perturbations in sepsis (Stare et al., 2015).
Keywords: Osmolality Osmolarity Serum osmolality Serum osmolarity Osmoregulation
Materials and Reagents
Underpad (AMG Medical, catalog number: 760-372 )
21 G x 1 ½ in. needle (BD, catalog number: 305167 )
5 ml syringe (BD, catalog number: 309646 )
1.5 ml microcentrifuge tube (Diamed, catalog number: DIATEC610-3169 )
1,000 μl pipette tips (Diamed, catalog number: DIATEC520-6753 )
Kimwipes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 06-666A )
1 rat (Rattus rattus; 100-300 g)
Isoflurane (CDMV, catalog number: 19417 )
100% oxygen
290 mOsm/kg control solution (ClinitrolTM 290 Reference Solution) (Advanced Instruments, catalog number: 3MA029 )
Equipment
Forceps (Dumont #7b forceps) (Fine Science Tools, catalog number: 11270-20 )
Veterinary gas anesthesia machine (Dispomed, model: 975-3300-000MRI )
Induction chamber (VetEquip, model: 941444 )
1,000 μl pipette (Gilson, PipetmanTM, model: F123602 )
Nosecone connected to Bain circuit (VetEquip, model: 921463 )
Centrifuge (Spectrafuge 24D) (Labnet International, model: C2400 )
Vortex (Vortex Genie 2) (Scientific Industries, model: SI-0236 )
Freezing point osmometer (Advanced Instruments, model: 3320 Micro-Osmometer )
Ease-Eject 20-Microliter sampler (Advanced Instruments, model: 3M0825 )
Sampler plunger wire (Advanced Instruments, catalog number: 3M0828 )
Sampler tips and chamber cleaners (Advanced Instruments, model: 3MA800 )
Procedure
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite： Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Stare, J. and Bourque, C. W. (2016). Measuring Rat Serum Osmolality by Freezing Point Osmometry. Bio-protocol 6(19): e1950. DOI: 10.21769/BioProtoc.1950.
Stare, J., Siami, S., Trudel, E., Prager-Khoutorsky, M., Sharshar, T. and Bourque, C. W. (2015). Effects of peritoneal sepsis on rat central osmoregulatory neurons mediating thirst and vasopressin release. J Neurosci 35(35): 12188-12197.
Download Citation in RIS Format
Category
Biochemistry > Other compound > Blood serum
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