flow cytometry is a technique that uses the principles of light scattering , light excitation , and fluorescence , to analyze size , complexity , molecular or cellular characteristics of single cells or particles by suspending them in a stream of fluid . this technique is widely used to analyze the expression of both extracellular and intracellular proteins , secreted molecules and dna - content , among others . it is also used in basic and clinical research ( immunology , hematology , molecular biology ) and recently in other research areas such as microbiology , algae , and plant biology . among the main advantages of flow cytometry are ( 1 ) the multi - parametric analysis of mixed populations by identifying specific markers on these cells and ( 2 ) the separation of particular subpopulations by electrical or mechanical means to divert cells with measured characteristics within the range specified by the user . they are large , meaning that their size exceeds that one of the host defense cells by several orders of magnitude . furthermore , the parasite and the host interphase is outside the cells , partially because most helminthes have a relatively impermeable cuticle composed of proteins that can be structurally arranged so they become  hard  . furthermore , it is worth noting that tissue dwelling nematodes poses a completely different problem than the better studied gastrointestinal nematodes . in the latter instances , live parasites can be successfully eliminated from within the mammal by such effectors as mucin of increased viscosity and enhanced peristaltic activity . such mechanisms are not available in the case of tissue dwelling nematodes , which have to be killed and perhaps broken down and digested before they can be successfully eliminated . thus , many host cells are literally covering all helminth body , as well as inside the parasite . this characteristic of helminthes makes it really difficult to isolate and characaterize antigens , or proteins that belong exclusively to the parasite , without host interference or contamination . thus , finding a method that is capable to render cells of only parasite origin is a challenge for researchers . for the past years , in our laboratory , we have been trying out to describe the role of sex steroids on different helminthes parasites . also , the specific receptors of the parasite able to respond to these molecules have been partially characterized [ 4 , 5 ] . previously , have been detected mrna and protein , respectively , for steroid hormone receptors by using real - time reverse - transcriptase polymerase chain reaction ( rt - pcr ) and fluorescent microscopy methods in several parasites . however , as previously mentioned , due to the extremely high interaction between helminthes and immune or other host cell types , which may eventually lead to host cell invasion into several parasitic tissues , it is critical to determine that the detected and analyzed protein(s ) are exclusively found in the helminth parasitic cells , and not as a consequence of host immune ( or any other type ) of cell contamination . thus , it was crucial to develop a method that could sort out very specifically , and without any possible error , the cross - contamination between host and parasite cells . to date , the available methods to dissect single parasite cells are quite expensive and complicated ( microscopic laser dissection , for instance ) . here we describe a method to dissect different helminthes cells , and by using flow cytometry , we were able to determine that there was no host cell contamination and provide information that demonstrate that , these cells are from only helminthes cell population based on both proportions and size of cells , as well as microscopic characterization . animal care and experimentation practices at the instituto de investigaciones biomdicas are frequently evaluated by the institute 's animal care and use committee , according to the official mexican regulations ( nom-062-zoo-1999 ) . mexican regulations are in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health ( nih and the weatherall report ) of the usa , to ensure compliance with established international regulations and guidelines . the protocol was approved by the committee on the ethics of animal experiments of the instituto de investigaciones biomdicas ( permit number : 2011  16 ) . mouse , rats , and pigs sacrifice to obtain parasites was performed under anesthesia , and all efforts were made to minimize animal suffering . taenia crassiceps cysticerci for each experimental session were obtained from intraperitoneally infected female mice and placed in tubes containing sterile pbs ( 1x ) supplemented with 100  u / ml of antibiotics - fungizone ( gibco , grand island ) . the tubes were centrifuged for 10  min at 1,500  rpm at 4c and the supernatants were discarded . the packed cysticerci were incubated in dmem serum - free medium ( sigma , st . louis , missouri ) . they were then centrifuged 3 times for 10  min at 1500  rpm for washing . after the final wash , the numbers of viable ( complete , translucent and motile cystic structures ) cysticerci were counted under a binocular microscope . ten viable nonbudding cysticerci of approximately 2  mm in diameter were selected and dispensed in 24-well culture plates ( falcon , bd labware , franklin lakes , new jersey ) in 1  ml dmem serum - free medium ( gibco brl ) and maintained at 37c under 5% co2 until used . once dissected , cysticerci were placed in tubes containing sterile pbs ( 1x ) supplemented with 100  u / ml of antibiotics - fungizone ( gibco , grand island , ny ) . the tubes were centrifuged for 10  min . , at 800  g at 4c , and the supernatant was discarded . the packed cysticerci were incubated in dmem serum - free medium ( gibco , brl , rockville , md ) . they were then centrifuged 3 times for 10  min at 800  g for washing . after the final wash , the numbers of viable ( complete and translucent cystic structures ) cysticerci were counted using a binocular microscope and placed in 24-well culture plates ( falcon , bd labware , franklin lakes , new jersey ) in 1  ml dmem serum - free medium ( gibco brl ) and maintained at 37c under 5% co2 until used . trichinella spiralis ( iss 406 ) was maintained in the laboratory by serial passage infections in balb / c mice and/or sprague - dawley rats . the infective - stage muscular larvae ( ml ) were recovered from experimentally infected mice at 30 days p.i . by a standard pepsin - hydrochloric acid digestion method . larvae were washed several times with pbs , and 4,000 ml were used to infect sprague - dawley rats . adult parasites were recovered from 1 to 6 days p.i . from the small intestine . the nbl ( 24  72  h old ) were obtained by incubating 4- , 5- , and 6-day - old adult parasites in rpmi medium ( gibco brl ) supplemented with 10% fbs ( hyclone ) and antibiotics ( gentamicin 50  mg / l and amphotericin b , sigma , 2  .5  mg t. crassiceps , t. solium and t. spiralis cells were extracted by tissue disruption according to the following protocols . t. crassiceps parasites were macerated using a nylon mesh  sandwich  ( 150  mm . meshes were washed with media , and cell suspension was centrifuged at 300  g for 5  min , and cells in pellet were recovered in pbs . t.   solium parasites were disaggregated initially by passing them through a 3  ml syringe to recover internal cells . cells recovered in rpmi media , centrifuged at 300  g for 5  min and pellets were recovered in pbs . t. spiralis larvae were disaggregated using a micropestle ( eppendorf , usa ) until no more clumps were visible . cells were centrifuged at 300  g for 5  min and cells in pellet recovered in pbs . t. crassiceps and t. spiralis larvae cells were stained with the following antibodies for 10  min at 4c : anti - mouse cd3-fitc , anti - mouse cd4 fitc , anti - mouse cd8-pe - cy5 , anti - mouse cd19-pe , anti - mouse mac-1 , and anti - mouse mac-3 ( all from bd biosciences ) , and washed with 500  l of staining buffer ( pbs ph.7.4 , 2% fetal bovine serum , 0  .02% nan2 ) . t. solium cysticerci - derived cells were stained with anti - human mhc - i - biotin antibody , washed once , and stained with apc - coupled streptavidin ( bd biosciences ) . all samples were analyzed by flow cytometry using an facs calibur ( bd , biosciences ) and data analyzed using the flowjo software . helmith - derived cells were fixed in 2% paraformaldehyde solution for 10  min at 37c and centrifuged at 300  g for 5  min . afterwards , they were incubated in absolute methanol for 30  min at 4c and centrifuged at 300  g for 5 minutes and then washed twice with 500  l of staining buffer ( pbs ph.7.4 , 2% fetal bovine serum , 0  .02% nan2 ) and resuspended in same buffer ( 100  l / test ) . t. crassiceps and t. solium cells were incubated in presence of mouse antiparamyosin ( 1  g / test ) ( kindly provided by dr . t. spiralis cells were incubated in the presence of mouse anticaveolin ( 1  g / test ) ( kindly provided by dr . guadalupe ortega - pierres , cinvestav , ipn . ) at room temperature for 20  min , and subsequently washed with 1  ml of staining buffer . cell pellets were resuspended separately in presence of the secondary antibody fitc or alexa488-conjugated goat anti - mouse ( zymed ) and incubated at 4c for 30  min in the dark . after this second incubation , cells were washed twice in staining buffer and centrifuged at 300  g for 5  min . cell pellets were resuspended in 500  l of staining buffer in absence of light and analyzed by flow cytometry using an facs calibur ( bd , biosciences ) . for dapi fluorescent staining , in vitro cultivated primary cells were collected , fixed in paraformaldehyde ( sigma ) for 10  min , and permeabilized with 0  .2% dapi at a concentration of 0  .5  g / ml was added to the fixed cells on the slide , followed by an incubation for 20  min in the dark . slides were then rinsed in pbs , fluoprep ( biomerieux ) was added , and a cover slip applied . , we used a nikon eclipse e600 microscope equipped with a nikon dxm1200  f ccd ( nikon corp . ; immunofluorescence staining experiments were carried out using a nikon eclipse 80  i microscope and a nikon dxm1200  c ccd ( nikon corp . ) . for dapi visualization , a 350  lp filter was used ( nikon , corp . ) . image processing and analysis was carried out using adobe photoshop cs3 ( adobe systems inc . , san jose , ca , usa ) and image pro plus 6  .2 ( media cybernetics , bethesda , md , usa ) . in figure 1 , it is clear that we were able to obtain isolated single cells of the cestodes ( a ) taenia solium , ( b ) taenia crassiceps , and the nematode ( c ) trichinella spiralis . the composition shown in the first photograph of every row shows the complete parasites , before of the isolation process . in the second picture of every row , the first step , by disrupting the parasites and , showing clumps of parasites , and , debris of pieces of the same . finally , in the third picture of every row , the isolated single cells of the three parasites are depicted . it is interesting to note that , taenia crassiceps ( b ) cells are multinucleated , and , due to the size ( 10  m ) it can not be a complete parasite . the same can be said also for taenia solium ( a ) and trichinella spiralis ( c ) . often , one may wish to monitor the presence or expression of several different molecules in cell culture . one component that is almost always monitored is the cell nucleus , which is stained with dapi , a molecular probe characterized by ex = 358  nm and em = 461  nm . dapi binds to the inner groove of dna present in cell nuclei and results in a blue emission that can be seen in the fluorescent microscope . staining of dapi in these parasite - isolated cells not only allows us to visualize the cell nuclei , but also allows an easy quantification of the number of cells in a given field of view . a representative example of the images processing steps and subsequent , phase contrast , counting of cell nuclei stained with dapi , and the overlay using both techniques are shown in figure 2 , where ( a ) is the composition to show t. solium cells , ( b ) t. crassiceps cells , and ( c ) t. spiralis cells . flow cytometry analysis firstly showed that ( b ) t. solium , ( c ) t. crassiceps and ( d ) t. spiralis cells were different in size and complexity from mouse spleen cells ( a ) . in fact , parasite cells were approximately 3-fold smaller and exhibited less complexity ( figures 3(b ) , 3(c ) , and 3(d ) ) than the mouse spleen cells ( figure 3(a ) ) . in addition , parasite cells showed no expression of the membrane markers cd3 , cd4 , cd8 which are typically present in some types of mammalian leukocytes or mhc i , marker of all types of mammalian cells ( figure 4 ) . on the other hand , in figure 5 , the facs analysis showed that t. solium isolated cells expressed calreticulin ( a ) and t. crassiceps isolated cells expressed paramyosin ( ag - b ) ( b ) , an exclusive component of the cytoskeleton of cestodes , nematodes , and insects , while the host cells did not showed expression of this molecule ( not shown ) . in the case of t. spiralis isolated single cells , there was a clear expression of caveolin-1 ( cloned , sequenced , and expressed exclusively of this parasite ) , while the host cells analyzed to look for the expression of this protein have a negative dying ( not shown ) . several methods are currently employed to evaluate expression of specific molecules in helminthes , including real - time reverse - transcriptase polymerase chain reaction ( real - time rt - pcr ) , western blot , double - dimension gels , and several microscopic assays . however , all these methods require homogenization of the whole helminth parasite cells , thereby preventing evaluation of individual cells or specific cell types in a given parasite tissue or area and can not differentiate from parasite cells and host cell contamination cells . the problem with analyzing helminths proteins is that helminth tissues are usually highly contaminated by host immune cells , thus preventing researchers to determine that the molecule they are trying to characterize truly belong to their parasites . our method of isolation of helminth cells is highly specific and is able to determine and sort specifically the parasite cells from the host cells . in addition , parasite cells can be sorted out and recovered alive to perform experiments in these isolated cells , like , in vitro culture , transfection , and possibly regeneration of complete parasites derived from single cells . the disaggregation , and separation for further flow cytometry analysis shown in here , is an important tool for determining protein expression of cell surface molecules and has also been shown to be useful for identifying expression of intracellular proteins in permeabilized cells . therefore , our method can be a potentially useful tool in the field of new protein helminth parasite discovery biology by providing a technique for analyzing all type of proteins from helminth parasite origin within intact parasite cells , with no host cell contamination . this type of analysis has not been previously reported and may pose increased difficulties due to nonspecific binding of antibodies to intracellular proteins . in addition to providing information on cells expressing specific proteins , once that we have the disaggregated parasites , the cells specifically detected as parasites cells , by size and granularity and by using a single specific protein marker ( as paramyosin and caveolin-1 in our case ) , are gated to use to determine the problem protein . this gives an indication of the relative amount of the searched protein expressed by an individual cell , thus providing a means to quantify that protein on each cell . another advantage of this technique is that expression of proteins of interest can be correlated with the degree of activation , maturation , or differentiation of given parasite cell types . finally , if complex mixtures of parasite helminthes cells are present ( as is the case ) , flow cytometry can be used to sort subpopulations of cells and , therefore , identify proteins expressed by specific parasite cell types . this method is , however , limited in its inability to specify the location within the cell ( nucleus , cytoplasm , mitochondria ) of intracellular proteins that are identified . specifically in the field of parasite hormone receptors , flow cytometry can be used to identify intracellular expression of steroid hormone receptors . we have been able to demonstrate and characterize expression of estrogen hormone receptors , from t. crassiceps   using real - time reverse - transcriptase polymerase chain reaction ( rt - pcr ) and fluorescent microscopy methods , we were able to determine that it was present , respectively , for this receptor in the whole parasite . this method could , therefore , be useful to answer a variety of scientific questions related to hormone receptors biology in the complex - host parasite interaction , including determining the responsiveness of specific cell types of parasites to steroid hormone treatment , for instance . this method would , therefore , be a very useful tool for rapid , high throughput measurement of not only hormone receptors , or receptors in general , but at the protein level in single , intact helminthes isolated cells . on the other hand , it was critical to determine that the obtained cell populations were exclusively found in the t. crassiceps and t. solium cysticercus , and t. spiralis larvae , and not a consequence of host immune , or other type of cell contamination , because , again , as shown elsewhere , there is extremely high interaction between parasites and host cells , which may eventually lead to host cell invasion into several parasitic tissues . for this reason , an alternative use of flow cytometry was employed to differentiate proteins from t. crassiceps , t. solium , and t. spiralis and their murine host by identifying exclusive molecules of the parasite , which are neither synthesized nor expressed by the host . this is the case of paramyosin , a muscle protein found only in invertebrates , such as drosophila melanogaster , caenorhabditis elegans [ 10 , 11 ] , taenia solium [ 12 , 13 ] and t. saginata , and caveolin-1  a protein that is implicated in t. spiralis differentiation . the flow cytometry studies showed that presence of the analyzed cells protein belonged specifically to the parasites , because paramyosin was only detected in t. crassiceps and t. solium cells , and caveolin-1 was only expressed in t. spiralis . in contrast , the nti - paramyosin or nti - caveolin-1 antibody did not recognize cells extracted from mouse , but they were positive for cd3 , cd4 , cd8 , cd19 , and macrophage antibodies , contrary to parasite cells . these results demonstrate that the analyzed parasite cells are in fact from the three parasite 's origin and not from other sources , and simultaneously accentuate the potential use of flow cytometry for differential identification of molecules from organisms with extremely close contact , such as helminthes parasites and their hosts .
in parasitology , particularly in helminthes studies , several methods have been used to look for the expression of specific molecules , such as rt - pcr , western blot , 2d - electrophoresis , and microscopy , among others . however , these methods require homogenization of the whole helminth parasite , preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ . also , the extremely high interaction between helminthes and host cells ( particularly immune cells ) is an important point to be considered . 
 it is really hard to obtain fresh parasites without host cell contamination . 
 then , it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin , and not a consequence of host cell contamination . 
 flow cytometry is a fluorescence - based technique used to evaluate the expression of extra - and intracellular proteins in different type cells , including protozoan parasites . 
 it also allows the isolation and recovery of single - cell populations . here 
 , we describe a method to isolate and obtain purified helminthes cells .