id
int64 7
5.24k
| url
stringlengths 48
63
| content
stringlengths 567
149k
|
|---|---|---|
10 | https://bio-protocol.org/exchange/protocoldetail?id=10&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Colony PCR Using Yeast Spheroplasted Cells
Bio-protocol Editor
Published: Jan 5, 2011
DOI: 10.21769/BioProtoc.10 Views: 14271
Download PDF
Ask a question
Favorite
Cited by
Abstract
The first part of this protocol involves the removal of yeast cell walls using the Zymolase enzyme. The resulting spheroplast cells can then be used as template for PCR. This quick and easy to implement protocol describes how to prepare spheroplasted yeast cells for colony PCR.
Materials and Reagents
Spheroplasted yeast cells
Zymolase 20 T [AMS Biotechnology (Europe)]
Sorbitol
Sodium phosphate
Zymolyase solution (see Recipes)
Equipment
Sterile pipette tips
Standard laboratory PCR machine
Wooden toothpicks
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Molecular Biology > DNA > PCR
Microbiology > Microbial genetics > DNA
Cell Biology > Cell structure > Cell surface
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
100 | https://bio-protocol.org/exchange/protocoldetail?id=100&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Scratch Wound Healing Assay
YC Yanling Chen
Published: Vol 2, Iss 5, Mar 5, 2012
DOI: 10.21769/BioProtoc.100 Views: 121829
Download PDF
Ask a question
How to cite
Favorite
Cited by
Abstract
The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer is created by scratching, and the “healing” of this gap by cell migration and growth towards the center of the gap is monitored and often quantitated. Factors that alter the motility and/or growth of the cells can lead to increased or decreased rate of “healing” of the gap (Lampugnani, 1999). This assay is simple, inexpensive, and experimental conditions can be easily adjusted for different purposes. The assay can also be used for a high-throughput screen platform if an automated system is used (Yarrow and Perlman, 2004).
Materials and Reagents
Human MDA-MB-231 cell line (ATCC, catalog number: HTB-26 ™)
Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
Ethanol (Sigma-Aldrich, catalog number: 459836 )
Crystal violet (Sigma-Aldrich, catalog number: C3886 )
Equipment
BD Falcon 24-well tissue culture plate (Fisher Scientific, catalog number: 08-772-1H; BD Biosciences, catalog number: 353226 )
Rainin pipet tips (1 ml) (Mettler-Toledo, catalog number: GPS-L1000 )
Cell culture incubator: 37 °C and 5% CO2
Software
Photoshop or ImageJ (http://rsb.info.nih.gov/ij/download.html)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, Y. (2012). Scratch Wound Healing Assay. Bio-protocol 2(5): e100. DOI: 10.21769/BioProtoc.100.
Download Citation in RIS Format
Category
Cancer Biology > Invasion & metastasis > Cell biology assays
Cell Biology > Cell movement > Cell migration
Cell Biology > Cell viability > Cell proliferation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,000 | https://bio-protocol.org/exchange/protocoldetail?id=1000&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
ImmunoFISH for Mice and Baboons Frozen Sections
FR Francesca Rossiello
MF Marzia Fumagalli
FF Fabrizio d’Adda di Fagagna
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1000 Views: 8845
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Apr 2012
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2012
Abstract
This protocol is optimized for immunoFISH staining of OCT section of mouse tissues. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) (Le et al., 2010) and FISH against telomeric DNA.
Keywords: ImmunoFISH DNA damage response Telomeres Baboon tissues
Materials and Reagents
Tissue
OCT
4% formaldehyde
PBS
Triton
Goat serum
BSA
Primary antibody: 53BP1 #NB 100-304 rabbit from Novus
Second antibody: goat anti-rabbit Alexa Fluor® 488 Dye
Triton
Glycine
Mowiol 4-88 reagent (Calbiochem®)
Formamide
Tris HCl, pH 7.4
Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
Tween-20
DAPI
Hybridization mixture (see Recipes)
Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
Wash solution I (see Recipes)
Wash solution II (see Recipes)
Equipment
Glass slide
Metal thermoblock
Humidified chamber
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cell Biology > Cell imaging > Fluorescence
Cell Biology > Tissue analysis > Tissue isolation
Biochemistry > Protein > Immunodetection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,001 | https://bio-protocol.org/exchange/protocoldetail?id=1001&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes
M Michael B. Francis
JS Joseph A. Sorg
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1001 Views: 10956
Original Research Article:
The authors used this protocol in May 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
May 2013
Abstract
Clostridium difficile is a Gram-positive, spore-forming, strict anaerobe and the leading cause of antibiotic-associated diarrhea (McFarland, 2008). Germination by C. difficile spores is the first step in pathogenesis. Thus, identifying mechanisms of C. difficile spore germination may lead to novel anti-germination therapies. This protocol describes a method for identifying germination-null phenotypes for C. difficile spores by introducing random mutations into actively growing C. difficile using ethyl methanesulfonate (EMS) as a mutagen (Francis et al., 2013).
Keywords: Clostridium difficile Germination Spore Mutagenesis
Materials and Reagents
A petri plate of viable C. difficile cells
Ethyl methanesulfonate (EMS)
Rifampin
Sterile water
Sucrose
10% (w/v) taurocholic acid
Thioglycollate
Lysozyme
Brain Heart Infusion (BD BactoTM, catalog number: 237500 )
Yeast Extract (BD DifcoTM, catalog number: 212750 )
0.1% (w/v) L-cysteine (Sigma-Aldrich, catalog number: C7352 )
BHIS medium (see Recipes)
BHIS agar medium (see Recipes)
Equipment
15 ml conical screw cap tubes
50 ml conical screw cap tubes
Anaerobic chamber
Spectrophotometer or microplate reader
Biosafety cabinet
Bench-top microcentrifuge
Swinging-bucket centrifuge
65 °C heat block
50 °C water bath
37 °C water bath
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 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:
Francis, M. B. and Sorg, J. A. (2013). EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes. Bio-protocol 3(24): e1001. DOI: 10.21769/BioProtoc.1001.
Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.
Download Citation in RIS Format
Category
Microbiology > Microbial genetics > Mutagenesis
Microbiology > Microbial cell biology > Cell isolation and culture
Molecular Biology > DNA > Mutagenesis
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,002 | https://bio-protocol.org/exchange/protocoldetail?id=1002&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Virulence Studies of Clostridium difficile
M Michael B. Francis
JS Joseph A. Sorg
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1002 Views: 7960
Original Research Article:
The authors used this protocol in May 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
May 2013
Abstract
Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) can colonize antibiotic-treated hosts (McFarland, 2008). Antibiotics alter the composition of the normal, benign microbial flora which leads to loss of colonization resistance (Wilson and Perini, 1988; Antonopoulos et al., 2009). C. difficile spores germinate to actively growing bacteria which secrete toxins that damage the colonic epithelium (Voth and Ballard, 2005). The use of animal models of C. difficile disease have allowed the identification of mechanisms of colonization and virulence factors (Lyras et al., 2009; Kuehne et al., 2010; Francis et al., 2013; Aubry et al., 2012; Carter et al., 2011). This protocol describes virulence studies of C. difficile in the hamster model of C. difficile infection (Bartlett et al., 1978; Sambol et al., 2001).
Keywords: Clostridium difficile Spore Virulence Hamster Animal model
Materials and Reagents
Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol
C. difficile spores
Female Syrian golden hamsters (80 g–120 g)
Clindamycin Injection, USP (150 mg/ml) (Hospira, catalog number: 0409-4052 )
Dulbecco’s Modified Eagle Medium (DMEM)
Equipment
Animal feeding needles (gavage needles)
1 ml syringe
Dedicated BSL2 animal facility
Scale
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbe-host interactions > In vivo model
Immunology > Host defense > General
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,003 | https://bio-protocol.org/exchange/protocoldetail?id=1003&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Preparation of Adult Mouse Muscle Stem Cells
JL Ju Li
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1003 Views: 10262
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Muscle stem cells are adult stem cells responsible for muscle development, growth and regeneration. Current knowledge suggests those cells are heterogeneous population shared their position between the sarcolemma and basal lamina of muscle fibers. This protocol describes the technique to dissociate and collect the stem cells from skeletal muscle of adult mice, and separate them from other cells found in muscle (e.g. fat, connective tissue). To purify and preserve those myofiber associated muscle stem cells, we use two steps of enzyme digestion followed by cell pre-plating procedures.
Materials and Reagents
Collagenase type II (Sigma-Aldrich, catalog number: C6885 )
Penicillin Streptomycin (Gibco®)
HEPES
Dulbecco’s Modified Eagle Medium (DMEM)
Dispase II (STEMCELL Technologies)
Bovine Growth Serum (Hyclone)
Collagenase type II solution (see Recipes)
Collagenase/Dispase solution (see Recipes)
Growth medium (see Recipes)
Equipment
Scissor
50 ml conical tube
Microscope
100 mm cell culture dish
40 μm filter
Fluorescence Activated Cell Sorter (FACS)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Li, J. (2013). Preparation of Adult Mouse Muscle Stem Cells. Bio-protocol 3(24): e1003. DOI: 10.21769/BioProtoc.1003.
Download Citation in RIS Format
Category
Stem Cell > Adult stem cell > Muscle stem cell
Cell Biology > Cell isolation and culture > Cell isolation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,004 | https://bio-protocol.org/exchange/protocoldetail?id=1004&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Influenza Virus-cell Fusion Inhibition Assay
Mayo Yasugi
Kazuyoshi Ikuta
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1004 Views: 10570
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
During viral infection to host cells, several viruses undergo the process of endocytosis and pH-dependent fusion. By fusion of viral membrane with host cellular membrane, the viral core invades to host cytoplasm. A part of monoclonal antibodies against viral membrane protein have potential to inhibit the viral fusion step. Here we describe in vitro influenza virus-cell fusion inhibition assay. The infected cells expressing viral membrane protein, such as hemagglutinin (HA), on cellular surface are incubated with monoclonal antibodies targeting viral membrane protein. Then the cells are incubated under low pH condition. If the antibody does not inhibit the fusion step, we can see multinucleated giant cells.
Keywords: Endocytosis PH-dependent fusion Viral entry Hemagglutinin Influenza virus
Materials and Reagents
Monkey kidney cell line CV-1 cells
MDCK-propagated Influenza B viruses (B/Florida/4/2006 and B/Malaysia/2506/2004) provided by the National Institute of Infectious Diseases, Japan
Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
Fetal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
Phosphate buffered saline without Ca2+ and Mg2+ (PBS)
Dulbecco’s Modified Eagle’s Medium F12 (DMEMF12) (Life Technologies, catalog number: 11320-033 )
Acetylated trypsin (Sigma-Aldrich, catalog number: T6763 )
Giemsa stain solution (Wako Pure Chemical Industries, catalog number: 079-04391 )
Methanol (Nacalai tesque, catalog number: 21915-93 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9576 )
Specific pathogen
Monoclonal antibody
MEM powder (Nisshin EM, catalog number: 05901 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
MES (Wako Pure Chemical Industries, catalog number: 344-08351 )
Fusion medium (see Recipes)
Equipment
96-well cell culture plate (Corning, catalog number: 3596 )
CO2 incubator (37 °C, 5% CO2)
Light microscope
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Host defense > General
Immunology > Antibody analysis > Antibody function
Cell Biology > Cell-based analysis > Cytosis
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
1 Q&A
hello! I had some questions during my experiments similar with you guys.
1 Answer
41 Views
May 13, 2024
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,005 | https://bio-protocol.org/exchange/protocoldetail?id=1005&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Isolation of Neutralizing Antibody
Mayo Yasugi
Kazuyoshi Ikuta
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1005 Views: 9536
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. Then each of prepared HuMAbs is confirmed whether it can neutralize the specific virus by in vitro neutralization assay.
Keywords: Influenza Hemagglutinin Monoclonal antibody Neutralization Vaccine
Materials and Reagents
Human blood immunized by vaccination or natural infection against specific pathogen
Human fusion partner SPYMEG cells (product of the Medical & Biological Laboratories Corporation, Ltd, Nagoya, Japan)
Madin-Darby canine kidney (MDCK) cells provided from RIKEN cell bank
MDCK-propagated Influenza B viruses (B/Florida/06/2004 and B/Malaysia/2506/2006) by the National Institute for Infectious Diseases, Japan
Phosphate-buffered saline without Ca2+ and Mg2+ (PBS)
HetaSep (STEMCELL Technologies, catalog number: 07906 )
Ficoll-Paque PLUS (GE, catalog number: 17-1440-03 )
Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, catalog number: 11995 )
Polyethylene glycol #1500 (PEG) (Roche Diagnostics, catalog number: 00-783-641-00 )
Fecal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
HAT supplement (Life Technologies, catalog number: 21060 )
Antibody against the specific pathogen
HT supplement (Life Technologies, catalog number: 11067 )
Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
Ethanol (Nacalai Tesque, catalog number: 14713-53 )
Hybridoma SFM (Life Technologies, catalog number: 12045-084 )
HiTrap Protein G HP (GE, catalog number: 17-0404-01 )
BM condimed (Roche Diagnostics, catalog number: 10-663-573-001 )
Heparin 5,000 units per 5 ml (Novo-Heparin, Mochida Phrrmaceutical)
FITC-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, catalog number: 109-096-097 )
DMEM-FBS medium (see Recipes)
DMEM-HAT medium (see Recipes)
DMEM-HT medium (see Recipes)
PBS (see Recipes)
Equipment
75 cm2 cell culture flask (T-75 flask) (IWAKI PUMPS, catalog number: 3110-075 )
15- and 50-ml plastic tube (BD Biosciences,, catalog number: 352096 and 352070 )
6- and 10-cm culture dish (IWAKI PUMPS, catalog number: 3010-060 and 3020-100 )
6-, 12-, 24-, 48- and 96-well cell culture plate (IWAKI PUMPS, catalog number: 3810-006 , 3815-012 , 3820-024 , 3830-048 and 3860-096 )
Light microscope
Fluorescent microscope
CO2 incubator (5% CO2, 37 °C)
Incubator
Peristaltic pump (Perista BioMini Pump; ATTO Corporation)
Slide A Lyzer Dialysis Cassetes (MW = 10 K) (Thermo Fisher Scientific, model: 2160728 )
Beaker
Stirrer
Stirrer bar
Millex-HV Filter Unit 0.45 μm (EMD Millipore, model: SLHVJ13SL )
2.5-ml syringe
Floater
21 G needle
Centrifuge (TOMY DIGITAL BIOLOGY, model: LC-121 ; TS-7 rotor )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 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:
Yasugi, M. and Ikuta, K. (2013). Isolation of Neutralizing Antibody. Bio-protocol 3(24): e1005. DOI: 10.21769/BioProtoc.1005.
Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.
Download Citation in RIS Format
Category
Immunology > Antibody analysis > Antibody function
Microbiology > Antimicrobial assay > Killing assay
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,006 | https://bio-protocol.org/exchange/protocoldetail?id=1006&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction
Mitsuhiro Nishimura
Kazuyoshi Ikuta
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1006 Views: 12828
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze the molecular interaction, enabling rapid and repetitive estimation with relatively small amount of sample. Here we describe a general protocol about SPR analysis on the interaction between viral antigen and human MAb (HuMAb) IgG. Anti-human Fcγ is first covalently crosslinked on the sensor chip by amine coupling, and then HuMAb of interest is immobilized via anti-Fcγ MAb IgG interaction as ligand. Antigen injected on the sensor chip causes the SPR change in time course as the result of association and dissociation. By analyzing the kinetics, association rate, dissociation rate, and dissociation constant are obtained.
Keywords: Molecular interaction Monoclonal antibody IgG Viral antigen Neutalization activity
Materials and Reagents
Purified HuMAb (IgG)
Purified antigen
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Nacalai tesque, catalog number: 17514-15 )
Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 198-13765 )
2 mol/L sodium hydroxide solution (Nacalai Tesque, catalog number: 37441-45 )
Sodium acetate, anhydrous (Wako Pure Chemical Industries, catalog number: 192-01075 )
Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA.2Na) (Dojindo Molecular Technologies, catalog number: 345-01865 )
Polyoxyethylene (20) sorbitan monolaurate (Wako Pure Chemical Industries, catalog number: 167-11515 )
Goat Anti-Human Fcγ (Jackson ImmunoResearch Laboratories INC., catalog number: 109-005-008 )
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific, catalog number: 22980 )
N-hydroxysulfosuccinimide (NHS) (Thermo Fisher Scientific, catalog number: 24500 )
Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
2-ethanolamine (Nacalai tesque, catalog number: 23405-42 )
Hydrochloric acid (Wako Pure Chemical Industries, catalog number: 080-01066 )
Magnesium chloride hexahydrate (Wako Pure Chemical Industries, catalog number: 135-00165 )
Sodium Chloride (Nacalai tesque, catalog number: 321319-45 )
Glycine (Nacalai tesque, catalog number: 17109-35 )
EDC/NHS solution
0.5 M HEPES buffer (pH 7.4) (see Recipes)
0.5 M EDTA (pH 8.0) (see Recipes)
20% (v/v) polysorbate 20 (see Recipes)
10x HBS-E (see Recipes)
1x HBS-EP (see Recipes)
400 mM EDC (see Recipes)
100 mM NHS (see Recipes)
1 M 2-ethanolamine (pH 8.5) (see Recipes)
50 mM NaOH (see Recipes)
10 mM Acetate buffer (pH 4.0, 4.5, 5.0, 5.5) (see Recipes)
Glycine buffer 10 mM (pH 1.8, 2.0 2.2, 2.4) (see Recipes)
1 M NaCl (see Recipes)
3 M MgCl2 (see Recipes)
Equipment
Instrument for SPR analysis, e.g. Biacore T200 (GE) or ProteOn XPR36 (Bio-Rad Laboratories)
Sensor chip (with carboxyl groups available for the amine coupling reaction), e.g. Series S Sensor Chip CM5 (GE, catalog number: BR-1005-30 ) for Biacore T200 or ProteOn GLM Sensor Chip (Bio-Rad Laboratories, catalog number: 176-5012 ) for ProteOn XPR36
Vacuum pump
0.22 μm PES PLUS bottom top filter (IWAKI PUMPS, catalog number: 8024-045 )
0.22 μm Millex-GV syringe filter unit (Millipore, catalog number: SLGV033RS )
20 ml syringe (Thermo Fisher Scientific, catalog number: SS-20ESZ )
Centrifuge
1.5 ml centrifuge tube (Ina-optika corporation, catalog number: CF-0150 )
Spectra/Por 3 Dialysis trial kit (SPECTRUM® LABORATORIES, catalog number: 132720T )
Stir bar and stirrer
UV spectrometer
pH meter
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 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:
Nishimura, M. and Ikuta, K. (2013). Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction. Bio-protocol 3(24): e1006. DOI: 10.21769/BioProtoc.1006.
Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.
Download Citation in RIS Format
Category
Immunology > Antibody analysis > Antibody-antigen interaction
Biochemistry > Protein > Interaction
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,007 | https://bio-protocol.org/exchange/protocoldetail?id=1007&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Measurement of Acetylcholine from Cell Lines
JL Jamie K. Lau
KB Kathleen C. Brown
Piyali Dasgupta
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1007 Views: 10261
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.
Materials and Reagents
A549 cells (American Type Culture Collection)
Human Epidermal Growth Factor (EGF) (Sigma Chemical, catalog number: E9644 )
100x Insulin Transferrin Selenium (ITS) (Life Technologies, catalog number: 41400-045 )
Rosewell Park Memorial Institute (RPMI) Medium -1640 (ATCC, catalog number: 41400-045)
50 μM Hydrocortisone (Sigma-Aldrich, catalog number: H6909 )
Boveine serum albumin (BSA) (US Biochem, catalog number: 10857 )
Disposable sterile tissue culture filters (Corning, catalog number: 431161 )
Choline/acetylcholine Quantification Kit (BioVision, catalog number: K615-100 )
Liquid nitrogen
Serum-Free RPMI (SF-RPMI) (see Recipes)
Neostigmine (Sigma Chemical, catalog number: N2001 ) (see Recipes)
Equipment
60 mm cell culture dishes (Corning, catalog number: 353002 )
Microfuge tube
ELISA Reader
Lyophilizer (Labonco)
Centrifuge
CO2 cell culture incubator
-80 °C freezer
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > Cellular energetics > Cell biology assays
Cell Biology > Cell metabolism > Other compound
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,008 | https://bio-protocol.org/exchange/protocoldetail?id=1008&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis
Sam-Geun Kong
M Masamitsu Wada
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1008 Views: 10141
Edited by: Ru Zhang
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, so that this protocol is useful for analysis of cp-actin dynamics that are induced by blue light.
Keywords: Actin Arabidopsis Blue light Chloroplast Organelle
Materials and Reagents
Plant materials
Arabidopsis thaliana transgenic plants expressing GFP-TALIN in wild-type or mutant plants (Kong et al., 2013). Actin probe lines such as LIFEACT-YFP and GFP-fABD2, THRUMIN1-GFP transgenic lines are also useful.
Young and fully expanded rosette leaves of 3-4-week-old plants grown under 100 μmol/m2/s white light (16 h)/dark (8 h) cycles at 23 °C.
Note: Plants grown on soil are better than those in the plate of MS medium. The reason is that the leaves of the plants grown in the plate are more susceptible to wither during sample handling than those grown on soil.
Red cellophane film (TokyoButaiShowmei, catalog number: No. 20 )
(Optional) 2,3-butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753 ) as an inhibitor of myosin ATPase that inhibits actin dynamics
Evacuation solution containing Silwet L-77 (Bristol-Myers Squibb Company, catalog number: BMS-SL7755 ) (see Recipes)
Equipment
Syringe (10 ml)
Confocal microscope (Leica Microsystems, model: SP5 equipped with a 63x/1.20 W objective lens and multi-line 100 mW argon laser)
Plant growth room or chamber
Red safe-light (red LED or fluorescent lamp filtered with red films)
Forceps
Razor blade
Scissors
A custom-made cuvette system and ring holder: this system is composed of two steel rings mating each other with complementary threads, two round cover glasses (22 mm in diameter, No. 1 and No. 5), and a silicon ring (100 μm in thickness) (Wada and Kong, 2011; see Video 1)
Software
Open source software ImageJ (http://rsbweb.nih.gov/ij/)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Kong, S. and Wada, M. (2013). Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis. Bio-protocol 3(24): e1008. DOI: 10.21769/BioProtoc.1008.
Download Citation in RIS Format
Category
Plant Science > Plant cell biology > Cell structure
Cell Biology > Cell imaging > Live-cell imaging
Biochemistry > Protein > Structure
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,009 | https://bio-protocol.org/exchange/protocoldetail?id=1009&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Colony Forming Assay for HCV-Replicon Cell Line
Chang Ho Lee
Seong-Wook Lee
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1009 Views: 9402
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Since the HCV genome is present exclusively in RNA form during replication, a number of anti-HCV drugs show appearance of rapid drug-resistant viruses. Therefore, it is important to test generation of drug-escape mutant viruses by developed antiviral drugs for their validity. Here, we describe a colony formation assay-based method to observe appearance of drug-resistant viruses against nucleic acid based anti-HCV drugs in genotype 1b based subgenomic replicon cell culture system (Lee et al., 2013).
Keywords: HEPATITIS C VIRUS REPLICON Colony forming assay
Materials and Reagents
Cell line: HCV-replicon Huh-7 human hepatoma cell line (HCV genotype 1b subgenomic replicon pFKI389neo/NS3–3’/5.1 containing the neomycin resistant gene, provided by R. Bartenschlager, Heidelberg University, German) (Lohmann et al., 1999; Krieger et al., 2001)
0.1% Trypsin-EDTA (WELGENE, catalog number: LS015-01 )
Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30919.03 )
100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, HycloneTM, catalog number: 3V30010 )
Lipofectamine 2000 (Life Technology, catalog number: 11668-019 )
NaCl (Sigma-Aldrich, catalog number: S5886 )
KCl (Sigma-Aldrich, catalog number: P5405 )
Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
KH2PO4 (Sigma-Aldrich, catalog number: P9791 )
NaOH (Sigma-Aldrich, catalog number: S5881 )
1x Phosphate Buffered Saline (PBS) (see Recipes)
Complete Dulbecco’s modified Eagle medium with high glucose (DMEM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30243.01 ) (see Recipes)
50 mg/ml G418 (Merck KGaA, catalog number: 345810 ) (see Recipes)
2% Paraformaldehyde solution (Sigma-Aldrich, catalog number: 158127 ) (see Recipes)
1% Methylene blue (Duksan Scientific, catalog number: MEE0-22002 ) (see Recipes)
Equipment
35 mm cell culture plate (Corning, catalog number: 430165 )
100 mm cell culture plate (BD Bioscience, catalog number: 353003 )
5% CO2, 37 °C Incubator (Thermo Fisher Scientific, catalog number: 311 )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lee, C. H. and Lee, S. (2013). Colony Forming Assay for HCV-Replicon Cell Line. Bio-protocol 3(24): e1009. DOI: 10.21769/BioProtoc.1009.
Download Citation in RIS Format
Category
Microbiology > Microbial genetics > Mutagenesis
Microbiology > Microbial cell biology > Cell viability
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
101 | https://bio-protocol.org/exchange/protocoldetail?id=101&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Tumor Cell Invasion Assay
YC Yanling Chen
Published: Feb 5, 2012
DOI: 10.21769/BioProtoc.101 Views: 39214
Download PDF
Ask a question
Favorite
Cited by
Abstract
Cell invasion assays have been used to study the interactions between tumor cells and the extracellular matrix (ECM), which not only provides a structural scaffold for the cell, but also contains various biological factors for the survival and growth of the cell. ECM gel contains the basic components of the ECM that provides a structural support for the cell to grow and move. Cells can secrete enzymes that degrade certain components of the ECM to move towards chemoattractants, or to simply establish niches for growth. Metastatic tumor cells often show more invasiveness to the ECM gel due to their higher motility and/or enzymatic activity for degrading ECM components. This protocol describes a tumor cell invasion assay to study the interactions between tumor cells and the ECM.
Materials and Reagents
ECM gel (Sigma-Aldrich, catalog number: E1270 )
Human MDA-MB-231 cell line (ATCC, catalog number: HTB-26 ™)
Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
Ethanol (Sigma-Aldrich, catalog number: 459836 )
Crystal violet (Sigma-Aldrich, catalog number: C3886 )
Equipment
Cell culture incubator: 37 °C and 5% CO2
Millicell cell culture inserts of 12 mm diameter 8 μm pores (Merck KGaA, catalog number: PI8P01250 )
Cotton swabs
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
Category
Cancer Biology > General technique > Cell biology assays
Cancer Biology > Invasion & metastasis > Cell biology assays
Cell Biology > Cell movement > Cell motility
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,010 | https://bio-protocol.org/exchange/protocoldetail?id=1010&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Intestinal Differentiation of Human ESCs
SO Soichiro Ogaki
SK Shoen Kume
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1010 Views: 7774
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from human ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from human ESCs. The protocol for human ESCs is principally very similar with that for the mouse ESCs, with some modifications.
Keywords: Intestine Embryonic stem cells differentiation endoderm human
Materials and Reagents
Human embryonic stem cells (ESCs)
M15 cells (ECACC, catalog number: 95102517 )
Mouse embryonic fibroblast (MEF)
0.1% gelatin
PBS
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco®, catalog number: 11995-075 )
DMEM low glucose (Gibco®, catalog number: 11885-084 )
KnockOut DMEM/F12 (Gibco®, catalog number: 12660-012 )
RPMI1640 (Gibco®, catalog number: 11875-093 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco®, catalog number: 11140-050 )
2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
KnockOut Serum Replacement (KSR) (Gibco®, catalog number: 10828 )
B27 supplements (Gibco®, catalog number: 17504044 )
Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
BIO (Calbiochem®, catalog number: 361550 )
DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
Y-27432 (Wako Pure Chemical Industries, catalog number: 688000 )
Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11 )
0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
EF medium (see Recipes)
hESC Medium (see Recipes)
EB medium (see Recipes)
hESCs Endoderm Medium (see Recipes)
Intestinal Medium (see Recipes)
Equipment
24 well plate (Corning, catalog number: 3526 )
10 mm dish (Corning, catalog number: 430167 )
60 mm dish (BD Biosciences, Falcon®, catalog number: 353004 )
Centrifuge
37 °C 5% CO2 Cell culture incubator
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Stem Cell > Embryonic stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,011 | https://bio-protocol.org/exchange/protocoldetail?id=1011&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Intestinal Differentiation of Mouse ESCs
SO Soichiro Ogaki
SK Shoen Kume
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1011 Views: 7937
Reviewed by: Salma HasanLin Fang
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from mouse ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from mouse ESCs.
Materials and Reagents
Mouse embryonic stem cells (ESCs)
M15 cells (ECACC: catalog number: 95102517 )
Mouse embryonic fibroblast (MEF)
0.1% gelatin
PBS
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco®, catalog number: 11995-075)
DMEM low glucose (Gibco®, catalog number: 11885-084 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco®, catalog number: 11140-050 )
2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
KnockOut Serum Replacement (KSR) (Gibco®, catalog number: 10828 )
Recombinant human LIF (Wako Pure Chemical Industries, catalog number: 129-05601 )
Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
BIO (Calbiochem®, catalog number: 361550 )
DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11)
0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
EF medium (see Recipes)
Maintenance Medium (see Recipes)
EB medium (see Recipes)
Endoderm Medium (see Recipes)
Intestinal Medium (see Recipes)
Equipment
6 well plate (Corning, catalog number: 3516)
100 mm dish (Corning, catalog number: 430167 )
60 mm dish (BD Biosciences, Falcon®, catalog number: 353004 )
Centrifuge
37 °C 5% CO2 Cell culture incubator
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Stem Cell > Embryonic stem cell > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell differentiation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,012 | https://bio-protocol.org/exchange/protocoldetail?id=1012&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)
XZ Xin Zhang
QL Qingsong Liu
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1012 Views: 27364
Edited by: Lin Fang
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three methods are frequently used to provide basic information about autophagy, people should keep in mind that they are not enough to give the exact details about the autophagy flux due to the complexity of this dynamic process. In addition, acridine orange assay is only a supplementary method to detect autophagy because it also has high affinity to other organelles such as lysosomes. For further investigation about a compound’s effect on autophagy flux, additional and more complicated assays are recommended. Protocols here provide a starting point for people to get a snapshot of whether a compound can affect autophagy in tissue culture cells.
Keywords: Autophagy LC3 Acridine orange Immunofluorescence Western blot
Materials and Reagents
70% ethanol
DMEM or other medium for tissue-culture cells
Bovine Serum Albumin (BSA) (Sigma-Aldrich)
Formaldehyde
100% Methanol
PBS
Rabbit antiLC3B antibody (LC3B (D11) XP® Rabbit mAb) (Cell Signaling Technology, catalog number: 3868 )
HRP-conjugated secondary antibody
Fluorescently labeled anti-rabbit secondary antibody (Molecular Probes, Life Technologies, catalog number: A-11008 )
ProLong® Gold Antifade Reagent with DAPI (Life Technologies, catalog number: P36935 )
M-PER lysis buffer (Pierce Antibodies, catalog number: 78501 )
5% Non-fat dry milk
cOmplete ULTRA Protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 0 5892970001 )
PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics, catalog number: 0 4906837001 )
Sample buffer (Bio-Rad Laboratories, catalog number: 161-0737 )
Enhanced chemiluminescence (ECL)
Acridine orange (Sigma-Aldrich, catalog number: A8097 )
4-15% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories, catalog number: 456-1085 )
E64d/pepstatin A (Sigma-Aldrich, catalog number: E8640/P5318 )
TBS (see Recipes)
TBS-Tween (see Recipes)
AbDil-Tw (see Recipes)
TBS-Tx (see Recipes)
AbDil-Tx (see Recipes)
Running buffer (see Recipes)
Transfer buffer (see Recipes)
Equipment
24-well plate
12-well plate (or 6-well plate)
1.5 ml tube
Round microscope coverslips (Fisher Scientific, catalog number: 12-545-81 ) (these coverslips are ready to use and do not require extra coating procedure)
Slides
Tweezer with fine tips
Transfer pipette
Humid chamber (can be homemade from a 15 cm plate, parafilm, foil and wet kimwipes)
Vacuum pump with a liquid bottle and pipe attached to remove washing solution
Fluorescent microscope
Western Blotting apparatus (SDS-PAGE running cassette, power supply, shaker, transfer cassette, PVDF membrane, etc.)
Tissue culture apparatus (CO2 incubator, tissue culture hood, etc.)
Software
MetaMorph
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Zhang, X. and Liu, Q. (2013). Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay). Bio-protocol 3(24): e1012. DOI: 10.21769/BioProtoc.1012.
Download Citation in RIS Format
Category
Cell Biology > Cell imaging > Fluorescence
Cell Biology > Cell signaling > Autophagy
Biochemistry > Protein > Immunodetection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,013 | https://bio-protocol.org/exchange/protocoldetail?id=1013&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Library Construction for Genome-wide Bisulfite Sequencing in Plants
DA David Moraga Amador
CW Chenggang Wang
KH Kevin H. Holland
ZM Zhonglin Mou
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1013 Views: 13886
Edited by: Feng Li
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
Materials and Reagents
DNA LoBind 1.5 and 2.0 ml centrifuge tubes (Eppendorf, catalog number: 22431021 and 22431048 , respectively)
AMPure magnetic beads (Beckman Coulter, catalog number: A63881 )
Ethanol (70% v/v, ethanol/water)
TruSeq DNA sample preparation kit (Illumina, catalog number: FC-121-2001 )
SYBR Safe (Life Technologies, catalog number: S33102 )
50 bp ladder (Thermo Fisher Scientific, catalog number: SM0373 )
Disposable sterile scalpels (Thermo Fisher Scientific catalog number 12-460--452 )
FalconTM 15 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 14-959-49B )
QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
Qubit dsDNA HS assay kit (Life Technologies, catalog number: Q32851 )
EpiTect bisulfite kit (QIAGEN, catalog number: 59104 )
MinElute PCR purification kit (QIAGEN, catalog number: 28004 )
PfuTurbo Cx hotstart DNA polymerase (Agilent, catalog number: 600410-51 )
KAPA library quantification kit (Kapa Biosystems, catalog number: KK4824 )
TE buffer (see Recipes)
Elution buffer (see Recipes)
Equipment
S2 adaptive focus acoustic disruptor (Covaris)
Microtubes with AFA fiber with pre-slit snap-caps (Covaris, catalog number: 520045 )
Refrigerated water bath circulator (Thermo Fisher Scientific, model: SC100-A10 )
Thermomixer R (Eppendorf, catalog number: 022670107 )
MiniSpin PlusTM centrifuge (Eppendorf, catalog number 0 22620100 )
Magnetic stand (Life Technologies, catalog number 4457858 )
Vortex mixer (Thermo Fisher Scientific, catalog number 02-215-365 )
2100 BioAnalyzer (Agilent, catalog number G2943CA )
Qubit 2.0 fluorometer (Life Technologies, catalog number Q32866 )
Horizon 11-14, horizontal gel electrophoresis system (Apogee Designs, model: 11068020 )
Safe Imager 2.0 blue light transilluminator (Life Technologies, model: G6600 )
ABI7900HT real-time PCR system (Life Technologies)
Thermal cycler (C1000 Touch) (Bio-Rad Laboratories, model: 185-1196EDU )
Pipettors (RAININ, various models)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Amador, D. M., Wang, C., Holland, K. H. and Mou, Z. (2013). Library Construction for Genome-wide Bisulfite Sequencing in Plants. Bio-protocol 3(24): e1013. DOI: 10.21769/BioProtoc.1013.
Download Citation in RIS Format
Category
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > DNA modification
Systems Biology > Epigenomics > DNA methylation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,014 | https://bio-protocol.org/exchange/protocoldetail?id=1014&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings
HY Haibing Yang
AM Angus Murphy
Published: Vol 3, Iss 24, Dec 20, 2013
DOI: 10.21769/BioProtoc.1014 Views: 24842
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including plasma membrane, intracellular vesicles, Golgi membranes, endoplasma reticulum, and tonoplast (vacuolar membrane) from 5-7 days old seedlings, which is often analyzed for auxin transporters in Arabidopsis (Leonard and Vanderwoude, 1976; Titapiwatanakun, et al., 2009; Yang et al., 2013; Blakeslee et al., 2007). After homogenization, plant debris including cell walls, chloroplasts and nucleus were removed by low speed centrifugation (8,000 x g), then total microsomal membranes were pelleted by high speed centrifugation (10,000 x g) and separated from soluble fractions. We secondly describe a method to separate microsomal fractions according to size or density in a sucrose density-gradient system by centrifugation. The linear sucrose gradient from 20%-55% (1.09-1.26 g cm-3) were used to separate membranes with different densities: tonoplast, 1.10-1.12 cm-3, Golgi membranes, 1.12-1.15 cm-3, rough endoplasmic reticulum 1.15-1.17 cm-3, thylakoids, 1.16-1.18 cm-3, plasma membrane, 1.14-1.17 g cm-3, and mitochondrial membranes, 1.18-1.20 cm-3 (Leonard and Vanderwoude, 1976; Larsson et al., 1987; Briskin and Leonard, 1980). However, the plasma membrane can also be isolated according to its outer surface properties which are very different from intracellular membrane surfaces. Thus, the right-side-out plasma membrane vesicles can be separated in an aqueous Dextran-polyethylene glycol two-phase system. The plasma membranes can be purified to > 90% in the upper phase (Larsson et al., 1987; Alexandersson et al., 2008). Two-phase systems for Arabidopsis seedlings were described in the section 3. Sucrose density gradient membrane fractionation followed by western blot is often used to analyze the distribution of certain membrane protein, while Two-phase separation is used when high purity of plasma membrane or intracellular membrane is required.
Keywords: Plant membrane preparation Sucrose density gradients Two-phase separation Arabidopsis
Materials and Reagents
Note: All chemicals were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com/united-states.html) unless otherwise specified.
Arabidopsis seedlings or mature tissue
Ice
Sucrose (Molecular Biology, Sigma-Aldrich, catalog number: 84097 )
HEPES (Sigma-Aldrich, catalog number: H-3375 )
EDTA (Research Products International, catalog number: E57020 )
PVP (40,000) (Fisher Scientific, catalog number: BP431 )
BSA (Sigma-Aldrich, catalog number: A-7906 )
DTT (Sigma-Aldrich, catalog number: D0632 )
Leupeptin (Sigma-Aldrich, catalog number: L2884 )
PMSF (Sigma-Aldrich, catalog number: P-7626 )
Benzamidine (Sigma-Aldrich, catalog number: B-6506 )
Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
Aprotinin (Sigma-Aldrich, catalog number: A1153 )
BTP-MES (BIS-TRIS propane, Sigma-Aldrich, catalog number: B6755 ; MES, Research Products International, catalog number: M22040 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Dextran (GE, catalog number: 17-0320-01 )
Polyethylene glycol 3350 (Union Carbide Corporation, catalog number: Carbowax 3350 )
EGTA (Sigma-Aldrich, catalog number: E3889 )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
Grinding buffer (see Recipes)
Resuspension buffer (see Recipes)
Stock solutions (see Recipes)
Phase mixture (see Recipes)
Phase system (see Recipes)
Equipment
Hermle Labnet Z383 centrifuge (Labnet International)
OptimaTM-L-90K ultracentrifuge (Beckman Coulter)
TLX centrifuge (TLA 100.3 rotor) (Beckman Coulter)
Waring blender jar (Waring Pro model: PBB212 )
Mortar and pestle
SW-28 rotor
Nylon SS-34 tube
SW-38 tube
3.5 ml polycarbonate TLA 100.3 tube
2 ml screwcap tubes
10, 15 and 50 ml Falcon tubes
Polyallomer ultracentrifuge tube (Beckman Coulter)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Yang, H. and Murphy, A. (2013). Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings. Bio-protocol 3(24): e1014. DOI: 10.21769/BioProtoc.1014.
Download Citation in RIS Format
Category
Plant Science > Plant biochemistry > Protein
Cell Biology > Organelle isolation > Membrane
Biochemistry > Protein > Isolation and purification
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,015 | https://bio-protocol.org/exchange/protocoldetail?id=1015&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti
HS Hongguang Shao
JL James B. Lok
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1015 Views: 14345
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Aug 2012
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Aug 2012
Abstract
Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm piggyBac transposon-mediated integration of transgenes into chromosomes of the parasitic nematode Strongyloides ratti. This work is described in detail in Shao et al. (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5’- and 3’ flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map piggyBac transposon-mediated chromosomal integrations in Drosophila, and was the source of our method for Strongyloides. The splinkerette- and piggyBac-specific oligos described in that reference could be used without modification in Strongyloides. For interested readers, a general review of the biology of parasitic nematodes in the genus Strongyloides may be found in Viney and Lok (2007), and a methods-based article on S. stercoralis as an experimental model, with information on transgenesis, may be found in Lok (2007).
Keywords: Strongyloides Transgenesis Chromosomal integration Transposon Splinkerette PCR
Figure 1. Diagrammatic representation of protocol for mapping transgene integrations in Strongyloides by splinkerette PCR (adapted from Potter and Luo, 2010)
Materials and Reagents
Free-living adult worms
Genomic DNA extraction
Gentra Puregene Tissue Kit (QIAGEN), including
Cell lysis solution (catalog number: 8304295 )
Protein precipitation solution (catalog number: 8273807 )
DNA hydration solution (catalog number: 8274043 )
Enzymes for digestion and treatment
Restriction enzymes include BstY I, BamH I and Bgl II (New England Biolabs)
Others include Shrimp Alkaline Phosphatase (United State Biological, catalog number: P4071-05 ) and Exonuclease I (New England Biolabs, catalog number: M0293S )
Ligation reagents
10x Ligase buffer and T4 Ligase (New England Biolabs, catalog number: M0202S )
PCR reagents
5x Phusion High-Fidelity Buffer, Phusion HF DNA Polymerase (New England Biolabs, catalog number: M0530S )
Oligonucleotides and primers
The oligonucleotides detailed in Table 1 must be synthesized
All sequences are from Potter and Luo (2010), Table S13
TE buffer (New England Biolabs, catalog number: E6293 )
10x NEB Buffer 2
1% agarose gel
Table 1. Oligonucleotides and primers for splinkerette PCR mapping of piggyBac transposon-mediated transgene integrations
Oligo or Primer
Sequence
SPLNK-BOT
5’-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGTGTCGACACTAGTGG-3’
SPLNK-GATC-TOP
5’-GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAA-3’
SPLNK#1
5’-CGAAGAGTAACCGTTGCTAGGAGAGACC-3’
SPLNK#2
5’-GTGGCTGAATGAGACTGGTGTCGAC-3’
3’SPLNK-PB#1
5’-GTTTGTTGAATTTATTATTAGTATGTAAG-3’
5’SPLNK-PB#1
5’-ACCGCATTGACAAGCACG-3’
3’SPLNK-PB#2
5’-CGATAAAACACATGCGTC-3’
5’SPLNK-PB#2
5’-CTCCAAGCGGCGACTGAG-3’
3’SPLNK-PB-SEQ
5’-ACGCATGATTATCTTTAAC-3’
5’SPLNK-PB-SEQ
5’-CGACTGAGATGTCCTAAATGC-3’
Equipment
PCR Thermal Cyclers (Mastercycler Personal, Eppendorf)
Gel electrophoresis
Gel documentation (FOTODYNE, model: FOTO/Analyst FX )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Shao, H. and Lok, J. B. (2014). Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti. Bio-protocol 4(1): e1015. DOI: 10.21769/BioProtoc.1015.
Shao, H., Li, X., Nolan, T. J., Massey, H. C., Jr., Pearce, E. J. and Lok, J. B. (2012). Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines. PLoS Pathog 8(8): e1002871.
Download Citation in RIS Format
Category
Microbiology > Microbial genetics > DNA
Molecular Biology > DNA > PCR
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,016 | https://bio-protocol.org/exchange/protocoldetail?id=1016&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
In vitro Nematode Infection on Potato Plant
Demosthenis Chronis
Shiyan Chen
Ping Lang
Tien Tran
DT David Thurston
Xiaohong Wang
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1016 Views: 13948
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Potato cyst nematodes (PCNs; Globodera rostochiensis and G. pallida) are devastating pests that infect potato root. We describe an in vitro assay for PCN infection on potato plantlet in tissue culture. This method is useful for studying nematode parasitism on potato and for investigating responses of potato clones/lines to PCN infection.
Keywords: Potato cyst nematode Globodera rostochiensis Nematode infection Potato plantlet
Materials and Reagents
Potato plant (Solanum tuberosum cv. Désirée)
Potato cyst nematode (G. rostochiensis) cysts
Sodium azide (RICCA Chemical, catalog number: 7144-16 )
Mercuric chloride (RICCA Chemical, catalog number: 4650-16 )
Sterile distilled water
Gentamycin (Fisher Scientific, catalog number: 61398-0010 )
Nystatin (Sigma-Aldrich, catalog number: N-3503 )
Agarose (Fisher Scientific, catalog number: BP160-100 )
Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
Micropore tape (Fisher Scientific, catalog number: 19-027-761 )
Sterilization solution (0.004% mercuric chloride/0.004% sodium azide)
Hoagland's solution (Sigma-Aldrich, catalog number: H2395 )
Amberlite XAD-4 resin (Fisher Scientific, catalog number: AC20223 )
MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
Inositol (Fisher Scientific, catalog number: AC122261000 )
Thiamine HCl (Sigma-Aldrich, catalog number: T1270 )
Gelrite (Fisher Scientific, catalog number: CAS 71010-52-1 )
Potato root diffusate (PRD) (see Recipes)
Propagation medium (see Recipes)
0.1% agarose (see Recipes)
Equipment
Sieves No. 60 (250 μm), No. 200 (75 μm) and No. 500 (25 μm) (Endecotts, catalog number: 683927 , 681837 , 691131 )
Sterile nematode egg hatching chamber (stainless steel metal pan cover and collection pan may be purchased from Vollrath Products, size six inches; the metal screened pan cover with wire-screen in the central area as shown in Figure 3 was custom made)
Sterile 6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
Stirplate
Sterile forceps and scalpel
Growth incubator
30-μm mesh (Small Parts, catalog number: 7050-1220-000-12 )
Tissue culture biosafety cabinet
Centrifuge
Incubator shaker (New Brunswick Scientific, model: C24KC )
S. tuberosum growth chambers (we use several types of growth chambers such as I-66LLVL from Percival)
Rubber stopper (size no. 1) (Fisher Scientific, catalog number: 14-130C )
Beaker
Aluminum foil
Dissecting microscope
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chronis, D., Chen, S., Lang, P., Tran, T., Thurston, D. and Wang, X. (2014). In vitro Nematode Infection on Potato Plant. Bio-protocol 4(1): e1016. DOI: 10.21769/BioProtoc.1016.
Download Citation in RIS Format
Category
Plant Science > Plant immunity > Disease bioassay
Cell Biology > Cell isolation and culture > Cell growth
Plant Science > Plant physiology > Plant growth
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,017 | https://bio-protocol.org/exchange/protocoldetail?id=1017&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Potato Transformation
Demosthenis Chronis
Shiyan Chen
Ping Lang
Tien Tran
DT David Thurston
Xiaohong Wang
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1017 Views: 18387
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
This is a protocol to produce stable transgenic potato plants (Solanum tuberosum cv. Désirée) by Agrobacterium-mediated genetic transformation, which is established based on a method described by (Jung et al., 2005) with some modifications. Agrobacterium tumefaciens strain LBA4404 carrying the desired construct is used to infect internodal explants to produce stable transgenic potato plants. Plantlet screening and molecular analyses are employed to confirm the expression of transgene in generated transgenic potato lines.
Keywords: Agrobacterium-mediated transformation Agrobacterium tumefaciens strain LBA4404 Potato plantlet
Materials and Reagents
Potato (S. tuberosum cv. Désirée) tissue culture plantlets
70% ethanol
Bleach (6% sodium hypochlorite)
Tween 20
Acetosyringone (Fisher Scientific, catalog number: AC11554 )
Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
Selective antibiotics
Mannitol (Fisher Scientific, catalog number: M120 )
Glycine
Nicotinic acid (Sigma-Aldrich, catalog number: N0761 )
Pyridoxine HCl (Sigma-Aldrich, catalog number: P6280 )
Thiamine HCl (Sigma-Aldrich, catalog number: T1270 )
Folic acid (Fisher Scientific, catalog number: BP2519 )
Biotin (Fisher Scientific, catalog number: BP232 )
MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
Inositol (Fisher Scientific, catalog number: AC122261000 )
Sucrose
6-benzylamino purine (BAP) (Sigma-Aldrich, catalog number: B3408 )
1-naphthalene-acetic acid (NAA) (Sigma-Aldrich, catalog number: N0640 )
3-indoleacetic acid (IAA) (Sigma-Aldrich, catalog number: I2886 )
Trans-zeatin-riboside (Sigma-Aldrich, catalog number: Z0876 )
YM medium (see Recipes)
MSVI vitamins (see Recipes)
JHMS vitamins (see Recipes)
3R vitamins (see Recipes)
CIM medium (see Recipes)
3C5ZR plates with selective agent (see Recipes)
Propagation medium (see Recipes)
TPS buffer (see Recipes)
Equipment
Sterile magenta boxes (sterilized by autoclaving)
Sterile petri dishes (Fisher scientific, catalog number: FB0875712 )
Sterile forceps and scalpel (sterilized by heat treatment using a Bunsen burner)
Sterile glass tubes with caps (sterilized by autoclaving) (tubes, Fisher Scientific, catalog number: 14-961-34 ; caps, Fisher Scientific, catalog number: 14-957-91D )
Sterile inoculating loop (Fisher Scientific, catalog number: 22-363-604 )
Tissue grinders
Glass culture tubes (sterilized by autoclaving)
3M Micropore tape (Fisher Scientific, catalog number: 1530-0 and 1530-1 )
Agrobacterium glycerol stock
50 ml conical centrifuge tubes
Tissue culture biosafety cabinet
Incubator shaker (New Brunswick Scientific, model: C24KC )
Centrifuge
S. tuberosum growth chambers (we use several types of growth chambers such as I-66LLVL from Percival)
Hot block or water bath
PCR thermal cycler (Bio-Rad Laboratories, model: C1000 touch)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chronis, D., Chen, S., Lang, P., Tran, T., Thurston, D. and Wang, X. (2014). Potato Transformation. Bio-protocol 4(1): e1017. DOI: 10.21769/BioProtoc.1017.
Download Citation in RIS Format
Category
Plant Science > Plant transformation > Agrobacterium
Molecular Biology > DNA > Transformation
Plant Science > Plant physiology > Tissue analysis
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,018 | https://bio-protocol.org/exchange/protocoldetail?id=1018&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Subcellular Localization Experiments and FRET-FLIM Measurements in Plants
ML Malgorzata Lichocka
ES Elmon Schmelzer
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1018 Views: 16934
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Determining the localization of proteins within living cells may be very essential for understanding their biological function. Usually for analysis of subcellular localization, a construct encoding the translational fusion of a cDNA of interest with a fluorescent protein (FP) is engineered, transiently expressed in plant cells and examined with confocal microscopy.
In co-localization and interaction studies, two plasmids, each encoding one of the potential interacting/binding partners tagged with an appropriate pair of fluorescence proteins (for instance CFP/YFP) are co-expressed in plant cells. If proteins co-localize in certain cellular compartments it does not necessarily mean that they bind/interact to each other, therefore an additional technique should be applied for in vivo verification of putative interaction, e.g. Fluorescence Lifetime Imaging (FLIM) to detect Fluorescence Resonance Energy Transfer (FRET).
The protocol describes in detail the method that has been used to verify interaction between the bacterial effector HopQ1 and a 14-3-3a host protein and additionally to check the necessity of the central serine in the canonical 14-3-3 binding site within HopQ1 (Giska et al., 2013) for this association.
Materials and Reagents
3 – 4 weeks old Nicotiana benthamiana plants grown in a greenhouse
Agrobacterium tumefaciens strain GV3101
For co-localization and FRET-FLIM measurement selected pGWB (Gateway) binary vectors were used (Nakagawa et al., 2007):
pGWB454 encoding Protein1 (here HopQ1-mRFP) -mRFP
pGWB441 encoding Protein2 (here 14-3-3a-YFP) -YFP
pGWB444 encoding unfused, free CFP
pGWB441 encoding unfused, free YFP
pGWB444 encoding CFP fused to YFP
pGWB441 encoding Protein1- (here HopQ1-YFP) -YFP
pGWB441 encoding Protein1a-YFP (e.g. mutated variant of Protein1)
pGWB444 encoding Protein2 (here 14-3-3a-CFP) -CFP
Equipment
For Microscopic evaluation
Nikon Stereomicroscope SMZ 1500 with epi-fluorescence equipment (optional)
Nikon Eclipse TE2000-E inverted C1 confocal laser scanning microscope, equipped with PlanApo 63x immersion oil objective, solid-state Coherent Sapphire 488-nm laser, Helium-Neon (HeNe) 543 nm laser, detector unit with three photomultiplier
Useful link, http://www.microscopyu.com/
For FRET-FLIM measurement
FRET-FLIM experimental procedure is optimized for a Zeiss LSM 510 META confocal laser scanning microscope equipped with a FLIM module SPC730 (Becker and Hickl) for time correlated single photon counting (TCSPC). Single photons were detected with a photomultiplier MCP-PMT, R3809U-52, Hamamatsu Photonics. For excitation of photons, an ultrafast oscillating multiphoton excitation laser (titanium-sapphire, Chameleon, Coherent) was used (Chameleon XS, Coherent)
Microscope slides (Gerhard Menzel GmbH, Menzel-GlaserTM, catalog number: AA00000112E )
Microscope Cover Slips no. 1 (Gerhard Menzel GmbH, Menzel-GlaserTM, catalog number: BB024060A1 )
Software
EZ C1 for Nikon C1 confocal – image acquisition
EZ C1 Viewer for Nikon C1 confocal – image analysis
LSM 510 for Zeiss Meta 510 Confocal – image acquisition
Zeiss LSM Image Browser – image analysis
SPC operation software for FLIM measurement (Becker & Hickl GmbH)
SPC-Image 2.9.1 software (Becker & Hickl GmbH, http://www.becker-hickl.de/pdf/flim-zeiss-man37.pdf) – image analysis
ImageJ freeware (http://rsbweb.nih.gov/ij/) – image analysis
MS Excel
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Lichocka, M. and Schmelzer, E. (2014). Subcellular Localization Experiments and FRET-FLIM Measurements in Plants. Bio-protocol 4(1): e1018. DOI: 10.21769/BioProtoc.1018.
Giska, F., Lichocka, M., Piechocki, M., Dadlez, M., Schmelzer, E., Hennig, J. and Krzymowska, M. (2013). Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiol 161(4): 2049-2061.
Download Citation in RIS Format
Category
Plant Science > Plant cell biology > Cell imaging
Cell Biology > Cell imaging > Fluorescence
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,019 | https://bio-protocol.org/exchange/protocoldetail?id=1019&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Biolistic Bombardment for Co-expression of Proteins Fused to YFP and mRFP in Leaf Epidermal Cells of Phaseolus vulgaris ‘Red Mexican’
ML Malgorzata Lichocka
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1019 Views: 12758
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
Biolistic bombardment is based on coating of tungsten or gold particles with DNA and delivery of these “biobullets” into living plant cells under high pressure (Sudowe and Reske-Kunz, 2013). This method enables transient expression of a DNA construct encoding fusion of the protein of interest to a fluorescence protein e.g. GFP for microscopic approaches. Usually it is performed for plants for which infiltration with Agrobacterium tumefaciens does not work efficiently e.g. model plant Arabidopsis thaliana (Ueki et al., 2009). Although transfection rate is relatively low, it is still sufficient to analyze subcellular localization of the protein of interest under a fluorescence microscope. Here we present the protocol that was optimized for Nicotiana benthamiana and also successfully applied to Phaseolus vulgaris (Giska et al., 2013).
Materials and Reagents
Upper leaves from 2 to 4 weeks old bean plants grown in a greenhouse
Plasmid DNA 2-4 μg
2.5 M CaCl2 (Sigma-Aldrich, catalog number: C3306-500G ) (optional other chlorides e.g. ZnCl2, MgCl2 can be used if it is necessary to avoid calcium ions)
0.1 mM Spermidine (Sigma-Aldrich, catalog number: S0266-1G )
75% , 96% and 100% ethanol
Sterile miliQ water
1 M spermidine stock solution (see Recipes)
0.1 mM spermidine working solution (see Recipes)
Equipment
Microcentrifuge (Eppendorf MiniSpin)
Rupture disks 1,100 psi (Bio-Rad Laboratories, catalog number: 165-2329 ) (available selection form 450 to 2,000 psi)
Microcarriers: Tungsten M17 (Bio-Rad Laboratories, catalog number: 165-2267 )
Macrocarriers (Bio-Rad Laboratories, catalog number: 165-2257 )
Stopping Screens (Bio-Rad Laboratories, catalog number: 165-2336 )
Vacuum pump (Bio-Rad Laboratories) (according to Bio-Rad technical recommendation)
Vortex
Forceps
1.5 ml microfuge tube (Eppendorf)
Parafilm
Petri dishes with wet filter paper at the bottom
Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories; www.bio-rad.com)
Stereomicroscope (Nikon Corporation, model: SMZ 1500 ) with epi-fluorescence equipment (optional)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant transformation > Bombardment
Plant Science > Plant cell biology > Cell imaging
Molecular Biology > DNA > Transformation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
102 | https://bio-protocol.org/exchange/protocoldetail?id=102&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Amplification of HIV-1 Infectious Virus in BL3 Lab
Xin Wang
Published: Vol 2, Iss 5, Mar 5, 2012
DOI: 10.21769/BioProtoc.102 Views: 12441
Download PDF
Ask a question
How to cite
Favorite
Cited by
Abstract
This method is used for making high titer human immunodeficiency virus type-1 (HIV-1) virus stock for subsequent infection assays. The amplification of T-tropic HIV-1 virus (IIIB strain) uses the CD4+ T cell line H9.
Materials and Reagents
CD4+ T cell line H9 cells (ATCC HTB-176 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH30071.03 HI )
Note: This particular FBS has been tested by the author, but may be substituted with FBS from different suppliers as desired by users.
Penicillin-Streptomycin liquid (Life Technologies, Gibco®, catalog number: 15070-063 )
Cell culture media: RPMI1640 (Life Technologies, Gibco®, catalog number: 11875-093 ) (see Recipes)
Equipment
Bench-top centrifuges
CO2 incubator
-80 °C freezer
50 ml conical tubes (BD Biosciences, Falcon®, catalog number: 35-2070 )
5, 10, 25-ml pipet (BD Biosciences, Falcon®, catalog number: 35-7501 , 35-7554 , 35-7556 )
T-75, T-175 cell culture flask (BD Biosciences, Falcon®, catalog number: 35-3136 , 35-3112 )
CryoTube Vials (NUNC, 377267)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Wang, X. (2012). Amplification of HIV-1 Infectious Virus in BL3 Lab. Bio-protocol 2(5): e102. DOI: 10.21769/BioProtoc.102.
Download Citation in RIS Format
Category
Microbiology > Microbe-host interactions > Virus
Microbiology > Microbial cell biology > Cell isolation and culture
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,020 | https://bio-protocol.org/exchange/protocoldetail?id=1020&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Cell Surface Protein-protein Binding on COS-7 Cells
KC Kae-Jiun Chang
MR Matthew N. Rasband
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1020 Views: 10410
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in May 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
May 2013
Abstract
Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.
Keywords: Membrane protein Protein-protein interaction Surface binding
Materials and Reagents
COS-7 cells (ATCC, catalog number: CRL-1651 TM)
A mammalian expression plasmid encoding a membrane protein of interest
A plasmid encoding an extracellular protein fused to the Fc portion of the human IgG [for example, pFUSE series by InvivoGen and pSXFc in Eshed et al. (2005) are options for cloning vectors to make this fusion construct]
Dulbecco's modified Eagle Medium (DMEM), high glucose, no glutamine (Life Technologies, catalog number: 11960-044 )
FetalClone III serum (Thermo Fisher Scientific, catalog number: SH30109.03 )
100x GlutaMAX-I (Life Technologies, catalog number: 35050-061 )
Lipofectamine 2000 (Life Technologies, catalog number: 11668-019 )
Dulbecco's phosphate-buffered saline (DPBS) (no calcium, no magnesium) (Life Technologies, catalog number: 14190-144 )
Virus production serum-free medium (VP-SFM) (Life Technologies, catalog number: 11681-020 )
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034 )
Protein Concentrators (9K MWCO, 7 ml) (Thermo Fisher Scientific, catalog number: 87748 )
Goat anti-human IgG, Fcγ fragment specific, conjugated with a fluorophore (e.g. Jackson ImmunoResearch Laboratories, catalog number: 109-545-098 for Fluorescein and 109-585-098 for Texas Red)
COS-7 culture medium (see Recipes)
0.2 M PB (see Recipes)
4% PFA (see Recipes)
PBS (see Recipes)
Equipment
6-well cell culture plates (Corning Incorporated, catalog number: 3516 )
Glass coverslips (VWR International, catalog number: 48366-227 )
10-cm dish
Centrifuge
37 °C/5% CO2 cell culture incubator
A regular fluorescence microscope
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chang, K. and Rasband, M. N. (2014). Cell Surface Protein-protein Binding on COS-7 Cells. Bio-protocol 4(1): e1020. DOI: 10.21769/BioProtoc.1020.
Download Citation in RIS Format
Category
Biochemistry > Protein > Immunodetection
Cell Biology > Cell structure > Cell surface
Molecular Biology > Protein > Protein-protein interaction
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,021 | https://bio-protocol.org/exchange/protocoldetail?id=1021&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
T Follicular Helper Cell Coculture Assay
RC Rafael A. Cubas
EH Elias K. Haddad
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1021 Views: 15737
Original Research Article:
The authors used this protocol in Apr 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Apr 2013
Abstract
T follicular helper (Tfh) cells constitute a distinct subset of CD4+ T cells specialized in providing help to B cells in germinal centers. Phenotypically, Tfh cells are characterized by their high expression of the chemokine receptor CXCR5 that allows their migration into B cell follicles as well as high expression of PD-1, BTLA, the co-stimulatory molecules ICOS and SLAM and the transcription factors BCL6 and cMaf. Tfh cells are the main producers of IL-21 as well as other cytokines like IL-4 and IL-10 critical for B cell survival and differentiation. Tfh cells drive somatic hypermutation and the generation of long-lived memory B cells and plasma cells having an essential role in the development of protective immunity. Developing a coculture system to measure the effects of Tfh-cell mediated B cell help is of great interest to further our understanding of Tfh-B cell interaction and to allow for the manipulation of culture conditions to investigate the potential effect different microenvironment signals or ligand/receptor interactions could have on Tfh cell function.
Materials and Reagents
Lymph node mononuclear cells (LMNCs)
Benzonse® Nuclease (EMD Millipore, catalog number: 70664 )
LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies, catalog number: L34957 )
Staphylococcal enterotoxin B (Toxin Technology Inc, catalog number: BT202 )
RPMI 1640 Medium 1x with L-Glutamine (Fischer Scientific, catalog number: 10-040-CV )
100x Penicillin-Streptomycin solution (5000 units) (Life Technologies, catalog number: 15070063 )
Fetal Bovine Serum (FBS) (Access Cell Culture)
RPMI 1640 Medium 1x with L-Glutamine and no Phenol red (Gibco®, Life Technologies, catalog number: 11835-030 )
HEPES solution (1 M) (Sigma-Aldrich, catalog number: H0887 )
Antibodies:
anti-CD3 (clone: HIT3a ) (BioLegend)
anti-CD4 (clone: RPA-T4 ) (BioLegend)
anti-CD45RA (clone: IM2711U ) (Beckman Coulter)
anti-CXCR5 (clone: RF8B2 ) (BD Biosciences)
anti-CD19 (clone: HIB19 ) (BioLegend)
anti-IgD (clone: IA6-2 ) (BD Biosciences)
anti-CD38 (clone: HIT2 ) (BioLegend)
Complete media (see Recipes)
Sorting buffer (see Recipes)
Equipment
Centrifuge (SorvallTM LegendTM XTR, Thermo Fisher Scientific)
15 ml Falcon tube
BD FACSAria II cell sorter (BD Biosciences)
37 °C 5% CO2 incubator
96-well sterile V-bottom plates (Thermo Fisher Scientific, catalog number: 249935 )
5 ml polystyrene round-bottom tubes with cell-strainer cap (BD FalconTM, catalog number: 352235 )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Cubas, R. A. and Haddad, E. K. (2014). T Follicular Helper Cell Coculture Assay. Bio-protocol 4(1): e1021. DOI: 10.21769/BioProtoc.1021.
Download Citation in RIS Format
Category
Immunology > Immune cell isolation > Lymphocyte
Cell Biology > Cell isolation and culture > Cell growth
Immunology > Immune cell function > Lymphocyte
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,022 | https://bio-protocol.org/exchange/protocoldetail?id=1022&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Agrobacterium-mediated Transformation of Strawberry
JS Jan G. Schaart
Published: Vol 4, Iss 1, Jan 5, 2014
DOI: 10.21769/BioProtoc.1022 Views: 13065
Edited by: Tie Liu
Original Research Article:
The authors used this protocol in Jan 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Jan 2013
Abstract
Traditional breeding for improvement of strawberry (Fragaria x ananassa) is difficult because strawberry is an octoploid, hybrid species. Genetic modification of strawberry would though be a promising alternative for obtaining the desired improvements in existing elite strawberry cultivars (Schaart et al., 2011). The availability of suitable genes for trait improvements in strawberry has however been a rate-limiting step until recently, but with the completion of the sequencing of the genome of woodland strawberry (F. vesca) (Shualev et al., 2011), we now have access to a treasure chest with valuable candidate genes. For strawberry, methods for genetic transformation have originally been described by Nehra et al. (1990) and James et al. (1990) and success of transformation was shown to be highly cultivar dependent. The latest progress in strawberry transformation is reviewed by Husaini et al. (2011). In our lab transformation of strawberry is based on the method for shoot regeneration described by Passey et al. (2003) and the use of the supervirulent Agrobacterium strain AGL0 (Lazo et al., 1991). We mainly make use of the strawberry transformation as a tool for functional analysis of candidate genes. For this the cultivar Calypso is a very suitable genotype because of its high transformation efficiencies (up to 100%) and ever-bearing fruiting characteristic, which provides a continuous supply of strawberry fruits once the plants start flowering.
Keywords: Strawberry Agrobacterium Transformation Genetic modification
Materials and Reagents
Sucrose (Duchefa Biochemie BV, catalog number: S0809 )
Daishin agar (Duchefa Biochemie BV, catalog number: D1004 )
6-Benzylaminopurine (BAP) (Duchefa Biochemie BV, catalog number: B0904 ) Indole-3-butyric acid (IBA) (Duchefa Biochemie BV, catalog number: I0902 )
Murashige and Skoog salts including vitamins (MS) (Duchefa Biochemie BV, catalog number: M0221 )
Gelrite (Duchefa Biochemie BV, catalog number: G1101 )
3’,5’Dimethoxy-4’-hydroxy-acetophenone (Acetosyringone) (Sigma-Aldrich, catalog number: D134406 )
LB broth (LB) (Sigma-Aldrich, catalog number: L3022 )
Rifampicin (Duchefa Biochemie BV, catalog number: R0146 )
Dimethyl sulfoxide (DMSO) (Merck KGaA, catalog number: 802912 )
MN615 filter paper 82 mm (MACHEREY-NAGEL GmbH & Co. KG, catalog number: MN615)
Thidiazuron (TDZ) (Duchefa Biochemie BV, catalog number: T0916 )
1-Naphthalene acetic acid (NAA) (Duchefa Biochemie BV, catalog number: N0903 )
Cefotaxime sodium salt (cefotaxime) (Duchefa Biochemie BV, catalog number: C0111 )
Kanamycin monosulphate monohydrate (kanamycin) (Duchefa Biochemie BV, catalog number: K0126 )
D-glucose monohydrate (glucose) (Duchefa Biochemie BV, catalog number: G0802 )
Shoot multiplication medium (SMM) (see Recipes)
Shoot propagation medium (SPM) (see Recipes)
Shoot regeneration medium (SRM) (gluc.) (see Recipes)
SRM + AS (see Recipes)
SRM SEL (see Recipes)
MS-liquid + AS (see Recipes)
1 mg/ml NAA (see Recipes)
0.22 mg/ml TDZ (see Recipes)
1 mg/ml IBA (see Recipes)
1 mg/ml BAP (see Recipes)
100 mM AS (see Recipes)
125 mM Cefotaxime (see Recipes)
50 mg/ml Kanamycin (see Recipes)
50 mg/ml Rifampicin (see Recipes)
30 g/100 ml Glucose (see Recipes)
Equipment
50 ml tube (Greiner Bio-One GmbH, catalog number: 210261 )
Petri dish (Greiner Bio-One GmbH, catalog number: 633180 )
Rotary shaker (C24 incubator shaker, New Brunswick Scientific)
Centrifuge Multifuge 3LR (Heraeus Holding)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Plant transformation > Agrobacterium
Molecular Biology > DNA > Transformation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,023 | https://bio-protocol.org/exchange/protocoldetail?id=1023&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Biochemical Assays for MTase Activity
Yu Chen
DG Deyin Guo
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1023 Views: 9793
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.
Keywords: Methyltransferase S-adenosyl-L-methionine RNA capping Cap structure
Materials and Reagents
Bodicon m7G capping system (Bodicon, catalog number: CS0130 )
S-adenosyl methionine (SAM) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
Bodicon Capping Enzyme (10 U/µl) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
Note: Because the sale of this kit was low, the previous companies which provided this capping kit were out of service. This capping kit was provided by a new company in China as custom-made products (contact e-mail: [email protected], phone:+86-13628662011). In fact the similar capping kit from any other companies (such as EPICENTRE biotechnologies, ScriptCap m7G capping system, catalog number: SCCE0610 ) is suitable for this experiment, and people can also contact with us to get the related protein or kit.
Inorganic pyrophospatase (YIPP) (New England Biolabs, catalog number: M2403S )
S-adenosyl [methyl-3H] methionine ([3H]-SAM) (PerkinElmer, catalog number: NET155H001MC )
DEAE Sephadex (GE Healthcare, catalog number: 17-0170-01 )
GTP (Thomas Scientific, catalog number: R0461 )
RNase inhibitor (Thomas Scientific, catalog number: EO0381 )
RNase free water
Phenol-chloroform (pH 4.8-5.2 for RNA only)
Ethanol (RNase free)
RNase free water
Sodium Dodecyl Sulfonate (SDS)
Ethylene Diamine Tetraacetic Acid (EDTA)
NH4HCO3
NaCl
10x MTase Buffer (see Recipes)
Cap-0 cap structure (m7GpppN-RNA) (see Recipes)
Non-methylated Cap-0 cap structure (GpppN-RNA) (see Recipes)
MTase assay reaction mix (see Recipes)
Equipment
Bechtop
Water bath
Centrifuge
Liquid scintillation counter
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Chen, Y. and Guo, D. (2014). Biochemical Assays for MTase Activity. Bio-protocol 4(2): e1023. DOI: 10.21769/BioProtoc.1023.
Download Citation in RIS Format
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Activity
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,024 | https://bio-protocol.org/exchange/protocoldetail?id=1024&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Monocular Deprivation in Mice
Ya-tang Li
XC Xiao-lin Chou
H Huizhong Whit Tao
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1024 Views: 9347
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Jul 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jul 2013
Abstract
Monocular deprivation is an experimental technique to study the ocular dominance plasticity during critical period (Hubel and Wiesel, 1963). Generally one eye of an animal is sutured during critical period, and the sutured eye is re-opened after either less than three days (short term) or more than three days (long term). Here we describe a detailed protocol for short-term and long-term monocular deprivation in mouse (Ma et al., 2013).
Materials and Reagents
Cotton
75% Ethanol
Ketamine hydrochloride (100 mg/ml) (Ketaject, Phoenix Pharmaceuticals, NDC number: 57319-542-02 )
Xylazine hydrochloride (100 mg/ml) (AnaSed Injection, LLOYD, NADA number: 139-236 )
Xylocaine (lidocaine hydrochloride) 2% Jelly (AstraZeneca, NDC number: 0186-0330-01 )
Bacitracin zinc ointment (USP 500 U/g) (Fougera Pharmaceuticals, NDC number: 0168-0011-31 )
Saline (0.9% sodium chloride injection USP)
Equipment
Hood
Autoclave
Aluminum foil
Dumont #5 forcep (Fine Science Tools, catalog number: 11254-20 )
Dumont #3c forcep (Fine Science Tools, catalog number: 11231-20 )
Cohan-Vannas spring scissor (Fine Science Tools, catalog number: 15000-02 )
Fine scissor (Fine Science Tools, catalog number: 14060-09 )
UNIFY Silk surgical suture [small (P-3) 13 mm, reverse cutting 3/8 circle needle. 4-0 18"/45 cm thread] (AD Surgical)
Alcohol pad (PDI, catalog number: B603 )
Gloves
Microscope
Illuminator (AmScope, catalog number: Model HL250-AY )
Heating pad (Harvard Apparatus, catalog number: 507220F )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Li, Y., Chou, X. and Tao, H. W. (2014). Monocular Deprivation in Mice. Bio-protocol 4(2): e1024. DOI: 10.21769/BioProtoc.1024.
Ma, W. P., Li, Y. T. and Tao, H. W. (2013). Downregulation of cortical inhibition mediates ocular dominance plasticity during the critical period. J Neurosci 33(27): 11276-11280.
Download Citation in RIS Format
Category
Neuroscience > Sensory and motor systems
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,025 | https://bio-protocol.org/exchange/protocoldetail?id=1025&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Preparation of Bacillus subtilis Cell Lysates and Membranes
JB Juri Niño Bach
MB Marc Bramkamp
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1025 Views: 15708
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
A common feature of every eukaryotic and prokaryotic cell is that they exhibit a plasma membrane. In Bacillus subtilis (B. subtilis) roughly 25% of all proteins are putative trans- or membrane associated proteins. Here we describe a relatively simple method to separate and prepare membrane and cytosolic proteins by ultra-centrifugation.
Materials and Reagents
Bacillus subtilis (B. subtilis)
Glycerol (Carl Roth, catalog number: 7530.4 )
Tris (J.T.Baker®, catalog number: 1414 )
NaCl (AppliChem GmbH, catalog number: A3597.5000 )
MgCl2 (Merck KGaA, catalog number: 1.05833.1000 )
Proteinase inhibitor (Roche Diagnostics, catalog number: 0 4693159001 )
DNAse I (Roche Diagnostics, catalog number: 10104159001 )
Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
Casein hydrolysate (Oxoid Limited, catalog number: LP0041 )
Buffer A (see Recipes)
Casein Hydrolysate (CH-medium) (see Recipes)
Solution G (see Recipes)
Equipment
Glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277 )
French press homogenizer (Glen Mills, French press G-MTM )
Ultra-centrifuge (Beckman Coulter, model: optimaTM XPN-100 )
Ti-70 Rotor (Beckman Coulter)
FastPrep tissue homogenizer (MP Biomedicals, model: 116004500 )
Refrigerated centrifuge (e.g. Beckman Coulter, model: Avanti-J25 )
Acrodisc® syringe filters (a pore size of 0.2 µm) (Pall, catalog number: 4652 )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Bach, J. N. and Bramkamp, M. (2014). Preparation of Bacillus subtilis Cell Lysates and Membranes. Bio-protocol 4(2): e1025. DOI: 10.21769/BioProtoc.1025.
Download Citation in RIS Format
Category
Cell Biology > Organelle isolation > Membrane
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,026 | https://bio-protocol.org/exchange/protocoldetail?id=1026&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Isolation of the Secretome from Bacillus subtilis
JB Juri Niño Bach
MB Marc Bramkamp
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1026 Views: 10622
Edited by: Fanglian He
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
Bacteria are commonly known to secret proteins in large amounts into the surrounding environment in high concentrations via various pathways. These proteins can be involved in numerous processes like cell-cell communication, exopolymer formation but also metabolic active enzymes are secreted that are interesting for industrial production of proteins. One of the most regularly used organisms for industrial protein production is the Gram-positive bacterium Bacillus subtilis (B. subtilis). Here we describe a protocol that can be used to quantitatively and qualitatively analyze secreted proteins from B. subtilis.
Materials and Reagents
B. subtilis
Trichloroacetic acid (Merck KGaA, catalog number: 1.00.807.0250 )
Acetone (J.T.Baker®, catalog number: 9006-01 )
BCA-assay (Pierce Antibodies, catalog number: 23225 )
Casein Hydrolysate (Oxoid Limited, catalog number: LP0041 )
(CH)-medium (see Recipes)
Solution G (see Recipes)
Resuspension Buffer (see Recipes)
Equipment
50 ml flasks with baffles
Acrodisc® syringe filters with a pore size of 0.2 µm (Pall, catalog number: 4652 )
Refrigerated centrifuge (e.g. Eppendorf, model: 5810R )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial biochemistry > Protein
Systems Biology > Proteomics > Secretome
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,027 | https://bio-protocol.org/exchange/protocoldetail?id=1027&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
A Protocol for Measurement of Intracellular pH
Iman Saramipoor Behbahan
Matthew A. McBrian
Siavash K. Kurdistani
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1027 Views: 22310
Edited by: Lin Fang
Reviewed by: Fanglian He
Original Research Article:
The authors used this protocol in Jan 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jan 2013
Abstract
Intracellular pH (pHi) is an important physiological determinant of enzyme activity and cellular function (Kurkdjian and Guern, 1989). All proteins depend on a tightly regulated pH to maintain their structure and function. Protonation–deprotonation events can dictate the charge of biological surfaces and are integral steps in many metabolic reactions (Casey et al., 2010). Moreover, the proton gradient across the mitochondrial membrane is used to generate cellular energy and support other mitochondrial processes. As a result, cells have developed multiple mechanisms to maintain a narrow range of pHi in response to extra- and intracellular fluctuations in pH (Orij et al., 2012). Here, we describe a protocol for pHi measurement in live cells that uses fluorescent microscopy and the pH sensitive dye 2’,7’-Bis-(2-Carboxyethyl)-5-(and-6-)-Carboxyfluorescein Acetoxymethyl Ester (BCECF-AM). This method was recently used to determine the effects of intracellular pH changes on global histone acetylation levels (McBrian et al., 2013).
Materials and Reagents
Hela cells
Nigericin (Sigma-Aldrich, catalog number: N7143 )
Prepare 10 mM nigericin (1:10,000 ) stock in ethanol (store aliquots at -20 °C and keep on ice during experiment)
DMSO (Sigma-Aldrich, catalog number: D2650 )
BCECF-AM (Life Technologies, catalog number: B1170 )
Glucose
1% Antibiotic-Antimycotic (Gibco®, catalog number: 15240 )
DMEM (Mediatech, Cellgro®, catalog number: 10-013-CV )
10% FBS
Paraffin wax
Calibration solution (see Recipes)
10x Earle’s balanced salt solution (EBSS) stock without glucose and without sodium bicarbonate (see Recipes)
Loading solution (see Recipes)
Cell culture medium (see Recipes)
Equipment
Perfusion inserts for 35 mm Dishes (Warner Instruments, catalog number: RC-33DM )
35 mm poly-lysine coated glass bottom culture dishes (MatTek, catalog number: P35GC-1.0-14-C )
50 ml conical tube
Needles
Aluminum foil
Axiovert 200 M Zeiss florescent microscope equipped with a high-resolution video camera (ZEISS, Axio CAM MRm)
BCECF filter set (Chroma Technology Corporation, catalog number: 71001a )
Neutralize density filter (ND 1.0 A - 10.0% transmission-25mm; this is custom-designed for a given microscope) (Chroma Technology Corporation)
Tubing and connections for perfusion
Flow pump (Rainin Peristaltic pump Dynamax RP-1. 4-channel; catalog number is different based on supplier)
5% CO2 tank
37 °C Water bath
5% CO2 incubator (make sure it is calibrated regularly; variations in CO2 concentration can have dramatic effects on pH, generating noise in the experiment)
Software
AxioVision 4.8
Slidebook 4.2
SigmaPlot
Excel or any spreadsheet analysis software
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Behbahan, I. S., McBrian, M. A. and Kurdistani, S. K. (2014). A Protocol for Measurement of Intracellular pH. Bio-protocol 4(2): e1027. DOI: 10.21769/BioProtoc.1027.
Download Citation in RIS Format
Category
Biochemistry > Other compound > Ion
Cell Biology > Cell-based analysis > Ion analysis
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,028 | https://bio-protocol.org/exchange/protocoldetail?id=1028&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Proteasome Assay in Cell Lysates
Pamela Maher
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1028 Views: 12559
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Jun 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Jun 2013
Abstract
The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.
Keywords: Nerve cells Fluorescence Protein degradation
Materials and Reagents
Cells
HEPES (Sigma-Aldrich, catalog number: H3375 )
MgCl2 (Sigma-Aldrich, catalog number: M2670 )
EDTA (Sigma-Aldrich, catalog number: E5134 )
EGTA (Sigma-Aldrich, catalog number: E4378 )
Sucrose (MP Biomedicals, catalog number: 821713 )
DTT (Life Technologies, catalog number: 15508013 )
Proteasome substrates [dissolved in DMSO (Sigma-Aldrich, catalog number: D8418 ) to a final concentration of 10 mM and stored frozen at -20 °C]
Suc-LLVY-AMC (chymotrypsin-like activity substrate) (Enzo Life Sciences, catalog number: P802 )
Z-ARR-AMC (trypsin-like activity substrate) (EMD Millipore, catalog number: 539149 )
Z-LLE-AMC (caspase-like activity substrate) (EMD Millipore, catalog number: 539141 )
PBS without Ca2+ and Mg2+ (Sigma-Aldrich, catalog number: P802)
Coomassie Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 1856209 )
BSA protein standard (Thermo Scientific, catalog number: 23209 )
ATP (Sigma-Aldrich, catalog number: A3377 )
Proteasome Lysis/Assay Buffer (see Recipes)
Equipment
60 mm tissue culture dishes
Rubber policeman or other type of cell scraper
1.7 ml microcentrifuge tube
Black walled 96 well plates (Corning, Costar®, catalog number: 3603 )
Clear 96 well plates (Greiner Bio-One GmbH, catalog number: 655101 )
Sonicator (GlobalSpec, model: W-380 )
Microcentrifuge
Fluorescent plate reader
Visible plate reader
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Maher, P. (2014). Proteasome Assay in Cell Lysates. Bio-protocol 4(2): e1028. DOI: 10.21769/BioProtoc.1028.
Valera, E., Dargusch, R., Maher, P. A. and Schubert, D. (2013). Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease. J Neurosci 33(25): 10512-10525.
Download Citation in RIS Format
Category
Neuroscience > Cellular mechanisms
Cell Biology > Cell imaging > Fluorescence
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,029 | https://bio-protocol.org/exchange/protocoldetail?id=1029&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
ADCC Assay Protocol
VS Vikram Srivastava
ZY Zheng Yang
IH Ivan Fan Ngai Hung
JX Jianqing Xu
BZ Bojian Zheng
MZ Mei-Yun Zhang
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1029 Views: 42236
Reviewed by: Lin FangFanglian He
Original Research Article:
The authors used this protocol in May 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
May 2013
Abstract
Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections and cancers in vivo. Here we developed a flow cytometry based protocol for ADCC assay using human peripheral blood mononuclear cells (PBMCs) as effector cells. Using this protocol, we determined the ADCC activity of convalescent plasma IgGs from six H1N1-infected human subjects in China, and identified two dominant ADCC epitopes, designated E1 [amino acid (AA) 92-117] and E2 (AA 124-159), on haemagglutinin of pandemic H1N1 influenza virus by epitope mapping of the convalescent plasma IgGs with different levels of ADCC activity. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer a comprehensive immune protection against viral infections.
Keywords: ADCC Influenza Virus Flowcytometry 7AAD Viability Staining Membrane labeling
Materials and Reagents
Raji cells (ATCC)
A/California/04/2009 (H1N1) influenza virus (Reference Laboratory)
Turkey red blood cells (from animal)
Tissue culture medium with serum (complete medium) RPMI 1640 (Life Technologies) containing 10% heat-inactivated fetal calf serum (FCS) and 2% L-glutamine
Serum, albumin, or other system-compatible protein (FBS) (Life Technologies, catalog number: 10099141 )
PKH67 cell labelling dye (Sigma-Aldrich, catalog number: MIDI67 )
7-AAD Stain (Life Technologies, catalog number: A1310 )
PBS (Calcium Magnesium free) (Life Technologies, catalog number: 10010-023 )
RPMI 1640 media (Life Technologies, catalog number: 12633-020 )
Pen/Strep (Life Technologies, catalog number: 10378016 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Equipment
Round bottomed 96-well plate
Temperature controlled centrifuge
Polypropylene conical bottom centrifuge tubes (4-15 ml)
Bio-safety Cabinet
Haemocytometer or cell counter
Slides and coverslips
Instrument (s) for fluorescence analysis (Flow cytometer)
37 °C, 5% CO2 incubator
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Srivastava, V., Yang, Z., Hung, I. F. N., Xu, J., Zheng, B. and Zhang, M. (2014). ADCC Assay Protocol. Bio-protocol 4(2): e1029. DOI: 10.21769/BioProtoc.1029.
Download Citation in RIS Format
Category
Immunology > Immune cell function > Cytotoxicity
Cell Biology > Cell-based analysis > Flow cytometry
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
103 | https://bio-protocol.org/exchange/protocoldetail?id=103&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Pulsed-field Gel Electrophoresis Typing of Gram-negative Bacteria (E.coli)
ZL Zhenying Liu
Published: Aug 5, 2011
DOI: 10.21769/BioProtoc.103 Views: 12975
Download PDF
Ask a question
Favorite
Cited by
Materials and Reagents
Chromosomal low melting agarose (Lonza InCert Agarose or Bio-Rad Laboratories)
Restriction enzymes (New England Biolabs)
Proteinase K (Promega Corporation)
10x Tris-borate EDTA (TBE) buffer (Promega Corporation)
Agarose for pulsed field gel (Bio-Rad Laboratories)
Lamda ladder for pulsed field gel (Bio-Rad Laboratories)
Ethidium bromide (10 mg/ml) (Life Technologies, Invitrogen™)
Agar media
TE
NaCl
EDTA
Sarkosy l
Sma I
Xba I
Restriction enzyme buffer
Running buffer (0.5x TBE)
Suspension buffer (see Recipes)
Lysis buffer (see Recipes)
Equipment
CHEF-MapperTM apparatus (Bio-Rad Laboratories)
Spectrophotometer (Bio-Rad Laboratories)
UV light and photograph
Water bath
Glass rod
1.5 ml Eppendorf tube
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Microbiology > Microbial genetics > DNA
Molecular Biology > DNA > Electrophoresis
Molecular Biology > DNA > DNA quantification
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,030 | https://bio-protocol.org/exchange/protocoldetail?id=1030&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Binding to Secreted Bone Matrix in vitro
AT Aurélie Jeanne Tormo
CB Christian Beauséjour
JG Jean-François Gauchat
Published: Vol 4, Iss 4, Feb 20, 2014
DOI: 10.21769/BioProtoc.1030 Views: 9146
Original Research Article:
The authors used this protocol in Mar 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Mar 2013
Abstract
This method examines the bone matrix binding capacity of proteins. Using osteogenic differentiation medium, multipotent stromal cells (MSC) are induced to differentiate into osteocytes in vitro and to secrete bone matrix. The latter is confirmed using Alizarin red S staining, which detects the presence of calcific deposits (hydroxyapatite). These calcific deposits are used to test the bone binding properties of proteins. The binding to the calcific deposits is assessed by Western blot analysis.
Materials and Reagents
Multipotent stromal cells (MSC) isolated from mouse bone marrow [see protocol “Isolation of Multipotent Stromal Cells from Mouse Bone Marrow” (Tormo et al., 2014)]
Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL )
Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL )
Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 12483 )
0.53 mM 0.05% Trypsin/EDTA (Wisent, catalog number: 325-042-EL )
PBS without Ca2+ and Mg2+ (Wisent, catalog number: 311-01-CL )
HEPES (Wisent, catalog number: 330-050-EL )
10 nM Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
50 μM Ascorbic acid-2 phosphate (vitamin C) (Sigma-Aldrich, catalog number: A4403 )
50 nM Cholecalciferol (Vitamin D3) (Sigma-Aldrich, catalog number: C9756 )
10 mM B-glycerophosphate (Sigma-Aldrich, catalog number: G9891 )
100x Protease and phosphatase inhibitors (Thermo Fisher Scientific, Pierce, catalog number: PI78447 )
IL-27 (1 μg) (R&D Systems, catalog number: 2799-ML-010/CF ) or any recombinant protein to be tested
Bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Pierce, catalog number: PI23225 )
Distilled water
Ethanol
Anti-IL-27 p28 biotinylated antibody (R&D Systems, catalog number: BAF1834 ) or biotinylated antibody specific for the protein to be tested
Streptavidin-HRP (Thermo Fisher Scientific, Pierce, catalog number: PI21130 )
SDS-PAGE loading buffer without bromophenol blue or DTT
Urea buffer (Fisher Scientific, catalog number: BP169-10 )
1 M Sodium phosphate monobasic (Fisher Scientific, catalog number: BP330-1 )
1 M Sodium phosphate dibasic (Fisher Scientific, catalog number: BP332-1 )
TBS-Tween
3% BSA
Dexamethasone solution (see Recipes)
10x Glycerophosphate solution (see Recipes)
1,000x Vitamin C solution (see Recipes)
Osteogenic differentiation medium (50 ml) (see Recipes)
Alizarin red S (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
Urea/Phosphate lysis buffer (see Recipes)
Equipment
15 ml tube
Centrifuge for cell culture
6 well tissue culture plates (BD Biosciences, catalog number: DL-353046 )
96 well tissue culture plates with flat bottoms (BD Biosciences, catalog number: DL-353072 )
Inverted phase-contrast microscope
37 °C, 5% CO2 cell culture incubator
Cell scrapers (Corning, catalog number: 3010 )
Microplate reader for BCA (562 nm)
SDS-PAGE Equipment
Western blot Equipment
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Biochemistry > Protein > Interaction
Cell Biology > Cell isolation and culture > Cell differentiation
Stem Cell > Adult stem cell > Stromal cell
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,031 | https://bio-protocol.org/exchange/protocoldetail?id=1031&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Isolation of Multipotent Stromal Cells from Mouse Bone Marrow
AT Aurélie Jeanne Tormo
MR Moutih Rafei
JG Jean-François Gauchat
Published: Vol 4, Iss 4, Feb 20, 2014
DOI: 10.21769/BioProtoc.1031 Views: 9311
Original Research Article:
The authors used this protocol in Mar 2013
Download PDF
Ask a question
Favorite
Cited by
Original research article
The authors used this protocol in:
Mar 2013
Abstract
Generating mouse multipotent stromal cells (MSC) from bone-marrow cells is usefull for a wide range of applications. Effectively, these MSC can differentiate into adipocytes, osteocytes [See “Binding to Secreted Bone Matrix in vitro” (Tormo et al., 2014)] or chondrocytes upon culture in specific differentiation medium.
Materials and Reagents
6-8 weeks old mouse
PBS without Ca2+ and Mg2+ (Wisent, catalog number: 311-01-CL )
Dulbecco's Modified Eagle's Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL )
Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 12483 )
Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL )
Trypan blue (Life Technologies, Gibco®, catalog number: 15250-061 )
Trypsin 0.05%/EDTA 0.53 mM (Wisent, catalog number: 325-042-EL )
APC-conjugated anti-CD31 antibody (clone MEC13.3) (BD Biosciences, catalog number: 551262 )
FITC-conjugated anti-CD45 antibody (clone 30-F11) (BD Biosciences, catalog number: 553080 )
APC-conjugated anti-CD44 antibody (clone IM7) (BD Biosciences, catalog number: 559250 )
PE-conjugated anti-CD105 antibody (clone MJ7/18) (BD Biosciences, catalog number: 562759 )
FITC-conjugated anti-CD90 antibody (clone 5E10) (BD Biosciences, catalog number: 555595 )
Equipment
Scissors and forceps
Syringe 1cc with 27 Gauge x 1-½ needle (BD, catalog numbers: BD-309659 and BD-305109 )
Petri dishes 100 x 20 mm (BD, catalog number: DL-353003 )
50 ml conical tubes (Progene®, catalog number: 71-5000-B )
Table top centrifuge
Culture hood
Hemocytometer
T-25 flask (BD, catalog number: 353108 )
37 °C, 5% CO2 Cell culture incubator
Flow cytometer (e.g. BD LSRFortessa)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell isolation > Maintenance and differentiation
Cell Biology > Cell isolation and culture > Cell isolation
Stem Cell > Adult stem cell > Stromal cell
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,032 | https://bio-protocol.org/exchange/protocoldetail?id=1032&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Retinal Explant Culture
CJ Christine Jolicoeur
Michel Cayouette
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1032 Views: 21049
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Nov 2012
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Nov 2012
Abstract
A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from embryonic animals and electroporated or infected with viral vectors, it is also a useful approach for the study of gene function at embryonic stages. Here, we present a retinal explant culture method that we have used extensively in various publications (Kechad et al., 2012; Cayouette et al., 2003; Cayouette and Raff, 2003; Elliott et al., 2008).
Keywords: Cell lineage Neural progenitor Asymmetric division Organotypic culture Differentiation
Materials and Reagents
Animals
We typically use retinas from albino Sprague Dawley rats or mice. Explants can be prepared from animals aged between embryonic day 12 to post-natal day 4.
DPBS (Life Technologies, catalog number: 14040 )
DMEM (Life Technologies, catalog number: 10569-010 )
Fetal bovine serum (FBS) (Wisent, catalog number: 080-450 )
Penicillin/streptomycin (Life Technologies, catalog number: 15070 )
70% ethanol
Fungizone antimycotic (Life Technologies, catalog number: 15290026 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
O.C.T. compound (Somagen, catalog number: 4583 )
Explant medium
4% Paraformadehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 ) (see Recipes)
20% sucrose/OCT (1:1) (see Recipes)
20% sucrose solution (see Recipes)
Equipment
Millicell organotypic insert (EMD Millipore, catalog number: PICMORG50 )
35 mm dish or 6 well plates
37 °C and 5% CO2 incubator
100 mm dish
150 mm dish
Straight forcep
FIne forcep (Fine Science Tools, catalog number: 11252-23 )
Curved forcep (Fine Science Tools, catalog number: 91197-00 )
Sharp scissor
Sharp scapel (Fine Science Tools, catalog number: 10315-12 )
Razor blade
P1000 pipette
CO2 chamber
Cryomold 15 x 15 x 5 mm (VWR International, catalog number: CA60872-016 )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Jolicoeur, C. and Cayouette, M. (2014). Retinal Explant Culture. Bio-protocol 4(2): e1032. DOI: 10.21769/BioProtoc.1032.
Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W., Harris, W. A. and Cayouette, M. (2012). Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina. J Neurosci 32(48): 17197-17210.
Download Citation in RIS Format
Category
Neuroscience > Development > Retinal culture
Cell Biology > Cell isolation and culture > Cell isolation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,033 | https://bio-protocol.org/exchange/protocoldetail?id=1033&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Dissociated Retinal Cell Culture
CJ Christine Jolicoeur
Michel Cayouette
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1033 Views: 15277
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Nov 2012
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Nov 2012
Abstract
The retina is a relatively simple and accessible part of the central nervous system, making it a powerful model to study cell fate specification mechanisms. Multipotent retinal progenitor cells (RPCs) give rise to seven major classes of retinal cell types. Mechanisms regulating cell fate choice in the retina depend on both cell intrinsic and environmental factors, but their relative contribution to specific cell fate decisions remains unclear. Dissociated retinal cell cultures provide a great assay to study this problem. RPCs are cultured in serum-free and extract-free medium, providing the investigator with a control over the environment to address questions related to the effects of a particular molecule on the development of retinal neurons. In addition, dissociated cell cultures can be used to study the importance of cell intrinsic mechanisms by isolating RPCs from their normal environment (Cayouette et al., 2003; Jensen and Raff, 1997). The method described below is suitable for the clonal-density culture of RPCs. In such cultures, RPCs are isolated from each other and from the postmitotic neurons. They divide and differentiate into different retinal cell types to form small colonies, or “clones”. In a recent study, we found that these clones are indistinguishable from the clones that develop in situ in the retina, both in terms of cell number and cell type composition, suggesting that intracellular mechanisms play a key role in retinal development (Cayouette et al., 2003).
Keywords: Retina Primary culture Retinal neuron Retinal progenitor cells Clonal density
Materials and Reagents
Animals
We typically use retinas from albino Sprague Dawley rats. This method works for culturing rat retinas aged between embryonic day 17 to post-natal day 1. Younger and older retinal cells or retinal cells from mouse retinas do not seem to survive as well under these conditions, but can also be cultured.
DPBS (Life Technologies, catalog number: 14040 )
Neurobasal medium (Life Technologies, catalog number: 21103-049 )
DMEM/F12 medium (Life Technologies, catalog number: 10565-018 )
B27 supplement (Life Technologies, catalog number: 17504-044 )
Pen/Strep (P/S) (Life Technologies, catalog number: 15140-122 )
Poly L-lysine (PLL) (Sigma-Aldrich, catalog number: P-4707 )
Mouse Laminin (Life Technologies, catalog number: 23017-015 )
Papain (Worthington Biochemical, catalog number: LS003126 )
L-cystein crystal (Sigma-Aldrich, catalog number: C-7352 )
Trypsin Inhibitor (Roche Diagnostics, catalog number: 109878 )
0.45 µm and 0.22 µm filters (Fisher Scientific, catalog number: SLHV033RS and SLGP033RS )
Hoechst 33342 (Life Technologies, Molecular Probes®, catalog number : H3570 )
“Incomplete” RGM medium
Growth factors (see Recipes)
bFGF (Pepro Tech, catalog number: 100-18B )
EGF (Pepro Tech, catalog number: 315-09 )
NT-3 (Pepro Tech, catalog number: 450-03 )
BDNF (Pepro Tech, catalog number: 450-02 )
10x Lo Ovomucoid (see Recipes)
20x 4% BSA (Sigma-Aldrich, catalog number: A-4161 ) (see Recipes)
0.4% DNase (Worthington Biochemical, catalog number: LS002007 ) (see Recipes)
Insulin (2.5 mg/ml) (Sigma-Aldrich, catalog number: I-6634 ) (see Recipes)
cpt-cAMP (Sigma-Aldrich, catalog number: C-3912 ) (see Recipes)
N-acetyl cysteine (NAC) (Sigma-Aldrich, catalog number: A-9165 ) (see Recipes)
Forskolin (Sigma-Aldrich, catalog number: D-2438 ) (see Recipes)
N2 Supplement (see Recipes)
4% Paraformaldehyde (Electron Microscopy Science, catalog number: 15710 ) (see Recipes)
Apo-Transferrin (Sigma-Aldrich, catalog number: T-1147 ) (see Recipes)
Progesterone (Sigma-Aldrich, catalog number: P-8783 ) (see Recipes)
Putrescine (Sigma-Aldrich, catalog number: P-5780 ) (see Recipes)
Sodium Selinite (Sigma-Aldrich, catalog number: S-5261 ) (see Recipes)
Equipment
35 mm petri dish (BD Biosciences, Falcon®, catalog number: 353001 )
50 ml conical tube
CO2 incubator
15 ml conical tube
100 mm petri dish
CO2 chamber
Straight forcep
Fine forcep (Fine Science Tools, catalog number: 11252-23 )
Curved forcep (Fine Science Tools, catalog number: 91197-00 )
Sharp scissor
Laminar flow hood
Water bath
P1000 pipette
Microscope
Hemacytometer
Centrifuge
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Jolicoeur, C. and Cayouette, M. (2014). Dissociated Retinal Cell Culture. Bio-protocol 4(2): e1033. DOI: 10.21769/BioProtoc.1033.
Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W. Y., Harris, W. A. and Cayouette, M. (2012). Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina. J Neurosci 32(48): 17197-17210.
Download Citation in RIS Format
Category
Neuroscience > Development > Retinal culture
Cell Biology > Cell isolation and culture > Cell isolation
Cell Biology > Tissue analysis > Tissue isolation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,034 | https://bio-protocol.org/exchange/protocoldetail?id=1034&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Monoclonal Antibody Purification (Nicotiana benthamiana Plants)
Adam Husk
Krystal Teasley Hamorsky
Nobuyuki Matoba
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1034 Views: 14278
Reviewed by: Pinchas Tsukerman
Original Research Article:
The authors used this protocol in May 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
May 2013
Abstract
Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of “deconstructed” virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a mAb per kg of leaf material in 7 days. A representative case for the broadly neutralizing anti-HIV and anti-influenza mAbs, VRC01 and CR6261 respectively, is shown (Hamorsky et al., 2013). Leaf tissue is homogenized and the extract is clarified by filtration and centrifugation. The mAb is purified by fast protein liquid chromatography (FPLC) using Protein A affinity and Phenyl HP hydrophobic interection resins.
Keywords: Monoclonal antibody Nicotiana benthamiana Plant virus vector
Materials and Reagents
Agrobacterium tumefaciens electrocompetent cells (strain GV3101)
MagnICON® plasmid (Icon Genetics GmbH, catalog number: pICH38099 ) (Marillonnet et al., 2004; Giritch et al., 2006)
Rifampicin (Sigma-Aldrich, catalog number: R3501 )
Gentamicin (Sigma-Aldrich, catalog number: G1264 )
Kanamycin (Sigma-Aldrich, catalog number: K1876 )
Yeast Extract (Fisher Scientific, catalog number: BP1422 )
Nutrient Broth (BD, catalog number: 231000 )
UV/Vis cuvette (VWR International, catalog number: 77776-745 )
MES (EMD Millipore, catalog number: 475894 )
Magnesium sulfate (EMD Millipore, catalog number: MX0070-3 )
Sodium phosphate (EMD Millipore, catalog number: SX0710-1 )
Sodium chloride (Sigma-Aldrich, catalog number: S9888 )
Sodium hydroxide
Ascorbic acid (Fisher Scientific, catalog number: BP351 )
1M Tris buffer (pH 8.0)
Glycine (Fisher Scientific, catalog number: BP381-5 )
L-Arginine (Sigma-Aldrich, catalog number: A5006 )
Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco®, catalog number: 14190-144 )
LB agar plate (see Recipes)
YenB media (see Recipes)
Infiltration buffer (see Recipes)
Extraction buffer (see Recipes)
Protein A Elution buffer (see Recipes)
Protein A Equilibration/Wash buffer (see Recipes)
Phenyl HP Equilibration/Wash buffer (see Recipes)
Phenyl HP Elution buffer (see Recipes )
Equipment
Amicon Ultra centrifugal filter, 30 K (EMD Millipore, catalog number: UFC903024 )
Electroporation cuvette (USA Scientific, catalog number: 9104-1050 )
Bottle top filter unit (VWR International, catalog number: 73520-986 )
Beveled flask (VWR International, catalog number: 4446-500 )
Centrifuge bottles (Thermo Fisher Scientific, catalog number: 3141-0250 )
HiTrap Protein A HP column (General Electric Company, catalog number: 17-0403-03 )
Phenyl HP column (General Electric Company, catalog number: 17-5195-01 )
Multiporator (Eppendorf, Bacteria module, catalog number: 4308 805.005 )
28 °C Incubator (Fisher Scientific)
Benchtop orbital shaker (Thermo Fisher Scientific, catalog number: SHKE4450 )
UV/Vis spectrophotometer (Beckman Coulter, catalog number: DU800 )
Avanti® J-26 XP Centrifuge (Beckman Coulter, catalog number: 393124 )
Infiltration apparatus (Bel-Art Products, catalog number: F420250000 )
Vacuum pump (William H. Welch Medical Library, catalog number: 8890A-75 )
Growth chamber
Blender (Waring Pro, catalog number: 7011HS )
AKTA purifier (General Electric Company, catalog number: 28-4062-66 )
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Husk, A., Hamorsky, K. T. and Matoba, N. (2014). Monoclonal Antibody Purification (Nicotiana benthamiana Plants). Bio-protocol 4(2): e1034. DOI: 10.21769/BioProtoc.1034.
Download Citation in RIS Format
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Isolation and purification
Immunology > Antibody analysis > Antibody detection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,035 | https://bio-protocol.org/exchange/protocoldetail?id=1035&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Neuron Culture from Mouse Superior Cervical Ganglion
MJ Marisa Jackson
WT Warren Tourtellotte
Published: Vol 4, Iss 2, Jan 20, 2014
DOI: 10.21769/BioProtoc.1035 Views: 13940
Edited by: Xuecai Ge
Original Research Article:
The authors used this protocol in Mar 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Mar 2013
Abstract
The rodent superior cervical ganglion (SCG) is a useful and readily accessible source of neurons for studying the mechanisms of sympathetic nervous system (SNS) development and growth in vitro. The sympathetic nervous system (SNS) of early postnatal animals undergoes a great deal of remodeling and development; thus, neurons taken from mice at this age are primed to re-grow and establish synaptic connections after in situ removal. The stereotypic location and size of the SCG make it ideal for rapid isolation and dissociation. The protocol described here details the requirements for the dissection, culture and differentiation of SCG neurons. The protocol is suitable for culturing neurons from late embryonic gestation to approximately postnatal day 3. The culture technique discussed below utilizes glass coverslips for the microscopic examination of fixed cells.
Materials and Reagents
Female mouse at desired gestational stage or early postnatal pups
Sterile PBS (Corning Cellgro®, catalog number: 21-040-CV )
L15 Leibovitz media (Corning Cellgro®, catalog number: 10-045-CV )
Collagenase, Type 4 (1 mg) (Worthington Biochemical, catalog number: LS004182 )
Collagenase enzyme solution (10 mg/ml in L15, filter-sterilized)
Trypsin, 0.25% EDTA, Mg2+ Ca2+-free (Corning Cellgro®, catalog number: 25-053-Cl )
Dulbecco’s Modified Eagle Medium (DMEM) (Corning Cellgro®, catalog number: 10-013-CV )
Fetal bovine serum (FBS) (Equitech-Bio)
Poly-D-lysine(PDL) (50 mg) (Sigma-Aldrich, catalog number: P0296 )
Boric acid (Sigma-Aldrich, catalog number: B7660 )
Sodium Tetraborate (Sigma-Aldrich, catalog number: B9876 )
Laminin (1 mg) (BD Biosciences, catalog number: 354232 )
2.5 s Nerve Growth Factor (100 μg) (BD Biosciences, catalog number: 356004 )
Concentrated nitric acid (Fisher Scientific, catalog number: A200-212 )
Penicillin/streptomycin mix (Life Technologies, catalog number: 15140-122 )
Sterile, deionized water
Cytosine arabinoside (AraC) (Sigma-Aldrich, catalog number: C6645 )
0.1 M borate buffer (pH 8.5) (see Recipes)
Regular plating medium (see Recipes)
Equipment
Tissue culture incubator
35 mm or 6-well TC plates
35 mm tissue culture treated Petri dishes
100 mm Petri dishes
150 mm plastic Petri dish
German glass coverslips, 25 mm (Electron Microscopy Sciences, catalog number: 72196-25 )
Ceramic racks (Thomas Scientific, catalog number: 8542E40 )
Basic gravity convection oven (VWR International, catalog number: 414005-108 )
Silicon rubber dissection plates
Scissors
Fine tipped forceps
26 gauge needles
Fire-polished, cotton-plugged, siliconized Pasteur pipets or Barrier tip 200
Reduced bore siliconized Pasteur pipets
Serological pipet
Stereoscopic microscope
Fume hood
Water bath
37 °C incubator
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 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:
Jackson, M. and Tourtellotte, W. (2014). Neuron Culture from Mouse Superior Cervical Ganglion. Bio-protocol 4(2): e1035. DOI: 10.21769/BioProtoc.1035.
Quach, D. H., Oliveira-Fernandes, M., Gruner, K. A. and Tourtellotte, W. G. (2013). A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation. J Neurosci 33(10): 4570-4583.
Download Citation in RIS Format
Category
Neuroscience > Development > Neuron
Neuroscience > Cellular mechanisms > Cell isolation and culture
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
1,036 | https://bio-protocol.org/exchange/protocoldetail?id=1036&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
In vitro Biomineralization Assay
Kyunghee Lee
Minsuk Kwon
Daewon Jeong
Published: Vol 4, Iss 3, Feb 5, 2014
DOI: 10.21769/BioProtoc.1036 Views: 18267
Original Research Article:
The authors used this protocol in Feb 2013
Download PDF
Ask a question
How to cite
Favorite
Cited by
Original research article
The authors used this protocol in:
Feb 2013
Abstract
Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including blood vessels, kidney, articular cartilage, and cardiovascular tissue. Here, we describe the simple method for detecting and measuring the presence of mineralized nodules in cardiac ventricular fibroblasts by using von Kossa and alizarin red S staining, and a colorimetric method for calcium quantification, respectively.
Keywords: Calcification Osteoblast Vascular smooth muscle cell
Materials and Reagents
Cardiac ventricular fibroblasts isolated from neonatal Sprague-Dawley rats by enzymatic dissociation (Lee et al., 2013)
Note: Our protocol can be used with various cell types such as osteoblasts and vascular smooth muscle cells.
Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and 4 mM L-glutamine (Hyclone, catalog number: SH 30243.01 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH 30919.03 )
Inorganic phosphate (Pi) (pH 7.4)
DPBS without Ca2+ and Mg2+ (Hyclone, catalog number: SH 30028.02 )
Trypsin/EDTA (Hyclone, catalog number: SH 30042.01 )
70% ethanol
Distilled deionized water (DDW)
0.6 N HCl solution
Lysis solution
0.1 N NaOH
0.1% sodium dodecyl sulphate (SDS)
QuantiChrome Calcium Assay Kit (BioAssay Systems, catalog number: DICA-500 )
Working reagent (refer to QuantiChrome Calcium Assay Kit manual for detail) (BioAssay Systems, catalog number: DICA-500) (see Reference 2)
Bio-Rad DC protein assay kit (Bio-Rad Laboratories, catalog number: 500-0016 )
Diluted protein standards (e.g., 0, 2, 4, 6, 8, 12, 16 and 20 mg/dl)
100 ng/ml receptor activator of NF-kB ligand (RANKL) (Sigma-Aldrich, catalog number: R0525 )
5% Aqueous silver nitrate solution (Sigma-Aldrich, catalog number: S7279 ) (see Recipes)
5% sodium thiosulfate (Sigma-Aldrich, catalog number: S7026 ) (see Recipes)
2% alizarin red S solution (Sigma-Aldrich, catalog number: A5533 ) (see Recipes)
1 M NaH2PO4 (pH 7.4) (Sigma-Aldrich, catalog number: S6566 ) (see Recipes)
Equipment
48-well plate
96-well plate
Spectrophotometer or 96-well reader
37 °C, 5% CO2 cell culture incubator
Inverted microscope
Aspirator
UV-visualizer
High-watt lamp (60-100 watt)
Procedure
Please login or sign up for free to view full text
Log in
/
Sign up
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite：Lee, K., Kwon, M. and Jeong, D. (2014). In vitro Biomineralization Assay. Bio-protocol 4(3): e1036. DOI: 10.21769/BioProtoc.1036.
Download Citation in RIS Format
Category
Immunology > Immune cell staining
Biochemistry > Other compound > Ion
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Write a clear, specific, and concise question. Don’t forget the question mark!
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Tags (0/5):
Post a Question
0 Q&A
Request a Protocol (RaP)
Request a detailed protocol from the "Materials and Methods" section of any research article.
Learn More
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
See all
About
About Us
Editors
Ambassadors
FAQs
Contact Us
For Preprint Authors
Submit a Protocol
Preprint Author Guidelines
Preprint Protocol Preparation Guidelines
For Advertisers
Advertising Integrity Board
Advertising Policy
Advertising Terms and Conditions
Other Resources
Bio-protocol Journal
Bio-protocol Preprint Repository
Bio-protocol Webinars
Request a Protocol (RaP)
© 2025 Bio-protocol LLC. ISSN: 2331-8325
Terms of Service Privacy Policy |
End of preview. Expand
in Dataset Viewer.
- Downloads last month
- 8