flow cytometry is a laser - based technology developed for the analysis of multiple physical and fluorescent parameters in cell populations . this technology works as suspended cells in a stream of fluid encounter a laser , exciting fluorescent markers on or within the cells . while flow cytometry has traditionally been used to identify populations of cells , it has proven to be a useful technology when studying an array of cell properties including cell membrane integrity , protein - protein interactions and protein trafficking . we have developed a protocol that allows this technology to be used to detect the state of chromatin in t cells as they are activated in vitro . we have also used this protocol to investigate the mechanism of activation - induced chromatin decondensation of t cells . t cells , a specific subset of lymphocytes within the immune system , are required for proper immune responses and the development of immunological memory . activation is initiated when an antigen is presented in the context of the major compatibility complex to the t cell receptor ( tcr ) located on the extracellular surface of quiescent t cells ( reviewed in ) . this triggers a series of dynamic and highly ordered molecular events within t cells that culminate in an increase in intracellular ca2 + concentration and the nuclear translocation of transcription factors required for activation ( reviewed in ) . once activated , t cells gain the ability to respond to interleukin-2 ( il-2 ) , a potent growth factor that utilizes the jak ( janus kinase)/stat ( signal transducer and activator of transcription ) pathway to drive clonal proliferation of activated t cells . briefly , il-2 stimulation results in the phosphorylation of stat proteins , a family of latent cytosolic transcription factors . once phosphorylated , stat proteins dimerize , translocate to the nucleus and drive expression of genes including those involved in cell cycle progression . in t cells , il-2 signals via stat5 , which is required for t cell proliferation . in order to achieve clonal expansion of activated t cells , those cells that have not experienced antigen - tcr engagement ( nave t cells ) , must have a mechanism to ignore the potent effects of il-2 . nave t cells possess a condensed chromatin that prohibits stat5-dna engagement in response to il-2 stimulation . upon activation , chromatin decondenses and stat5 can access the promoters of target genes , permitting clonal proliferation . interestingly , this change in chromatin status is not dependent on epigenetic modification of histone proteins ( for review , see ) as we observed no global change in histone modification during t cell activation .   while performing these studies , we discovered that antibodies raised against histone proteins also had difficulty accessing their epitopes in nave cells , but that upon activation could more readily bind their epitopes . here we present the method to use flow cytometry to detect fluorescently conjugated histone h3 antibodies in order to assess chromatin status in t cells . chromatin condensation in a population of cells is measured as the mean florescence intensity ( mfi ) of histone h3 staining . in the context of t cell activation , the mfi of histone h3 staining increases , signifying the decondensation of chromatin . in addition to measuring chromatin status via intracellular histone h3 staining , this protocol also incorporates surface staining and a fixable stain for live cells , permitting analysis of subpopulations of cells . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . the animal protocol was approved by the furman university institutional animal care and use committee ( permit number : a3242 - 01 ) . all materials and equipment used in this protocol can be found in the table of materials and equipment , while buffers used can be found in the table of buffers and solutions . note : use spleens from two 6 - 8 week old isogenic mice ( e.g. ,  c57b/6 ) of the same gender . typically , two spleens are processed . spray the animals profusely with 70% ethanol solution and orient such that the head is facing left . using forceps , lift the skin of the animal upwards and away from the body . using scissors , cut a small notch through the skin near the abdomen of the animal . using fingers , pull each side of the notch to expose the peritoneum from the neck to the beginning of the hind legs . the spleen should be visible directly underneath the peritoneum . using forceps , lift the peritoneum and make a small incision to expose the spleen . remove the spleen with forceps and use scissors to tease away any adipose and connective tissue . place the spleen into a 50 ml conical tube containing 10 ml pbs supplemented with 2% fetal bovine serum ( hereafter referred to as pbs+2% ) . note : typical recovery is between 60 - 90 million cells per spleen , and typically , 2 million cells are needed per sample . obtain two frosted microscope slides and hold the slides so that the two rough frosted surfaces face inwards towards one another . hold the spleen between the frosted surfaces of the slides , with the edges still submerged , and grind the spleen gently by moving the slides back and forth against one another ( figure 1a ) . continue grinding until all cells have been released and the remains of the spleen appear white ( figure 1b ) . draw up the cell suspension in a pipet and filter it slowly through a 70 m filter into a new sterile 50 ml conical tube . note that small pieces of red pulp will be present on the filter ( figure 2a ) . if the filter rises up , gently lift it up slightly to allow the fluid to drain through and place the filter back into the 50 ml conical tube and continue , repeating this process as necessary . if processing more than 5 spleens , it may be necessary to divide into two batches and use a new filter for each . if the filter rises up , gently lift it up slightly to allow the fluid to drain through and place the filter back into the 50 ml conical tube and continue , repeating this process as necessary . if processing more than 5 spleens , it may be necessary to divide into two batches and use a new filter for each . using the stopper portion of a 3 ml syringe gently press the red pulp containing the remaining cells through the strainer ( figure 2b ) . make sure to press both the filter bottom and sides to ensure that all red pulp has passed through the filter ( figure 2c ) . add 10 mlof pbs + 2% to the tissue culture dish and use this to wash the slides , syringe stopper , and dish to recover any remaining cells . pass this through the same 70 m cell strainer into the 50 ml conical tube . centrifuge the filtered cell suspension at 300 x g for 5 min at 4 c . lyse red blood cells by adding 2 ml of ack lysis buffer ( 0.15 m nh4cl , 1 mm khco3 , 0.1 mm edta , ph to 7.2 , store at 4 c ) per spleen to the conical tube and rotate and invert the tube to ensure that all cells come into contact with the ack buffer . gently swirl the tube for 1 min at rt . bring the volume of the cell suspension to 50 ml by adding pbs + 2% the tube to neutralize the ack buffer . invert the tube 10 times and centrifuge for 5 min at 300 x g at 4 c . after gently decant and discard the supernatant , careful not to disturb the pellet . leave a trace amount of pbs + 2% in the tube , cap the tube , and flick the pellet to resuspend it . add 10 ml of t cell media [ 10% fbs , 10 mm hepes ( ph 7.0 ) , 2 mm glutamax , 1 mm sodium pyruvate , 1x non - essential amino acids , 1x penicillin / streptomycin and 50 mm -mercaptoethanol ] , and further resuspend the pellet by gently pipetting up and down . then filter the suspension through a new 70 m filter into a new 50 ml conical tube . use another 5 - 10 ml of t cell media to rinse the original tube and pass this through the 70 m filter into the tube containing the rest of the filtered cells . if processing more than two spleens , the volume of t cell media can be increased to maximize recovery . cells should be counted and viability accessed . to count cells , combine 3 ml of cells with 24 ml of pbs + 2% and 3 ml of 0.4% trypan blue . viability , as assessed by trypan blue exclusion , is typically between 85 - 95% . 
  to count cells , combine 3 ml of cells with 24 ml of pbs + 2% and 3 ml of 0.4% trypan blue . viability , as assessed by trypan blue exclusion , is typically between 85 - 95% . resuspend cells in t cell media to a concentration of 1 x 10 cells / ml . transfer 2 million cells ( 100 ml ) into a well of a u remove the supernatant from each well by flicking liquid within the well plate into the sink ( the cell pellet will remain in the well ) and dabbing the plate on a clean paper towel . wash the cells by resuspending them in 200 l pbs followed by centrifugation at 300 x g for 10 min at 4 c . perform all washes this way unless otherwise noted . note : do not use pbs + 2% as it will interfere with the fixable pi stain . remove the supernatant by flicking the plate and add 100 ml of freshly prepared commercial stain ( e.g. , fixable red dead cell stain ) to each well . make the stain by diluting the reactive dye stock solution 1:10 in dmso followed by a 1:10 dilution in pbs . make the stain by diluting the reactive dye stock solution 1:10 in dmso followed by a 1:10 dilution in pbs . centrifuge the plate for 10 min at 300 x g at 4 c . at this point forward , remove the supernatant by flicking the plate and then add 100 ml of fc block solution . dilute antibodies to be used for surface staining ( e.g. ,  anti - cd4 or anti - cd8 ) in pbs and add 100 ml to each well . note : surface stain antibodies should be titered for optimal performance . typically use a 1:200 or 1:400 dilution , per the manufacturer s protocol . following incubation of the surface stain , wash the cells twice in pbs . after the last wash , add 100 l of 4% paraformaldehyde to each well . make perm / block solution ( stock perm solution is pbs + 2% + 0.02% triton x-100 , store at 4 c ) by combining 2 ml of normal rabbit serum per 100 ml stock perm solution . add 40 ml perm / block to each sample well and mix well by gently pipetting up and down , taking care not to make bubbles . dilute fluorescently conjugated histone h3k4me1 antibody in perm / block solution reserved in step 3.5 and add 10 l of this dilution to each well . note : use an h3k4me1 antibody conjugated using the r - phycoerythrin conjugation kit following the manufacturer s instructions . resuspend the cells in 200 l pbs+2% and transfer the samples to facs tubes for flow cytometry analysis . confirm euthanasia by cervical dislocation . note : use spleens from two 6 - 8 week old isogenic mice ( e.g. ,  c57b/6 ) of the same gender . spray the animals profusely with 70% ethanol solution and orient such that the head is facing left . using forceps , lift the skin of the animal upwards and away from the body . using scissors , cut a small notch through the skin near the abdomen of the animal . using fingers , pull each side of the notch to expose the peritoneum from the neck to the beginning of the hind legs . the spleen should be visible directly underneath the peritoneum . using forceps , lift the peritoneum and make a small incision to expose the spleen . remove the spleen with forceps and use scissors to tease away any adipose and connective tissue . place the spleen into a 50 ml conical tube containing 10 ml pbs supplemented with 2% fetal bovine serum ( hereafter referred to as pbs+2% ) . note : typical recovery is between 60 - 90 million cells per spleen , and typically , 2 million cells are needed per sample . obtain two frosted microscope slides and hold the slides so that the two rough frosted surfaces face inwards towards one another . hold the spleen between the frosted surfaces of the slides , with the edges still submerged , and grind the spleen gently by moving the slides back and forth against one another ( figure 1a ) . continue grinding until all cells have been released and the remains of the spleen appear white ( figure 1b ) . draw up the cell suspension in a pipet and filter it slowly through a 70 m filter into a new sterile 50 ml conical tube . note that small pieces of red pulp will be present on the filter ( figure 2a ) . if the filter rises up , gently lift it up slightly to allow the fluid to drain through and place the filter back into the 50 ml conical tube and continue , repeating this process as necessary . if processing more than 5 spleens , it may be necessary to divide into two batches and use a new filter for each . if the filter rises up , gently lift it up slightly to allow the fluid to drain through and place the filter back into the 50 ml conical tube and continue , repeating this process as necessary . if processing more than 5 spleens , it may be necessary to divide into two batches and use a new filter for each . using the stopper portion of a 3 ml syringe gently press the red pulp containing the remaining cells through the strainer ( figure 2b ) make sure to press both the filter bottom and sides to ensure that all red pulp has passed through the filter ( figure 2c ) . add 10 mlof pbs + 2% to the tissue culture dish and use this to wash the slides , syringe stopper , and dish to recover any remaining cells . pass this through the same 70 m cell strainer into the 50 ml conical tube . centrifuge the filtered cell suspension at 300 x g for 5 min at 4 c . lyse red blood cells by adding 2 ml of ack lysis buffer ( 0.15 m nh4cl , 1 mm khco3 , 0.1 mm edta , ph to 7.2 , store at 4 c ) per spleen to the conical tube and rotate and invert the tube to ensure that all cells come into contact with the ack buffer . gently swirl the tube for 1 min at rt . bring the volume of the cell suspension to 50 ml by adding pbs + 2% the tube to neutralize the ack buffer . invert the tube 10 times and centrifuge for 5 min at 300 x g at 4 c . after gently decant and discard the supernatant , careful not to disturb the pellet . leave a trace amount of pbs + 2% in the tube , cap the tube , and flick the pellet to resuspend it . add 10 ml of t cell media [ 10% fbs , 10 mm hepes ( ph 7.0 ) , 2 mm glutamax , 1 mm sodium pyruvate , 1x non - essential amino acids , 1x penicillin / streptomycin and 50 mm -mercaptoethanol ] , and further resuspend the pellet by gently pipetting up and down . then filter the suspension through a new 70 m filter into a new 50 ml conical tube . use another 5 - 10 ml of t cell media to rinse the original tube and pass this through the 70 m filter into the tube containing the rest of the filtered cells . if processing more than two spleens , the volume of t cell media can be increased to maximize recovery . cells should be counted and viability accessed . to count cells , combine 3 ml of cells with 24 ml of pbs + 2% and 3 ml of 0.4% trypan blue . viability , as assessed by trypan blue exclusion , is typically between 85 - 95% . 
  to count cells , combine 3 ml of cells with 24 ml of pbs + 2% and 3 ml of 0.4% trypan blue . viability , as assessed by trypan blue exclusion , is typically between 85 - 95% . resuspend cells in t cell media to a concentration of 1 x 10 cells / ml . transfer 2 million cells ( 100 ml ) into a well of a u remove the supernatant from each well by flicking liquid within the well plate into the sink ( the cell pellet will remain in the well ) and dabbing the plate on a clean paper towel . wash the cells by resuspending them in 200 l pbs followed by centrifugation at 300 x g for 10 min at 4 c . perform all washes this way unless otherwise noted . note : do not use pbs + 2% as it will interfere with the fixable pi stain . remove the supernatant by flicking the plate and add 100 ml of freshly prepared commercial stain ( e.g. , fixable red dead cell stain ) to each well . make the stain by diluting the reactive dye stock solution 1:10 in dmso followed by a 1:10 dilution in pbs . make the stain by diluting the reactive dye stock solution 1:10 in dmso followed by a 1:10 dilution in pbs . centrifuge the plate for 10 min at 300 x g at 4 c . at this point forward , perform all work with the plate with the tissue culture hood light off . remove the supernatant by flicking the plate and then add 100 ml of fc block solution . dilute antibodies to be used for surface staining ( e.g. ,  anti - cd4 or anti - cd8 ) in pbs and add 100 ml to each well . note : surface stain antibodies should be titered for optimal performance . typically use a 1:200 or 1:400 dilution , per the manufacturer s protocol . following incubation of the surface stain , wash the cells twice in pbs . after the last wash , add 100 l of 4% paraformaldehyde to each well . make perm / block solution ( stock perm solution is pbs + 2% + 0.02% triton x-100 , store at 4 c ) by combining 2 ml of normal rabbit serum per 100 ml stock perm solution . add 40 ml perm / block to each sample well and mix well by gently pipetting up and down , taking care not to make bubbles . dilute fluorescently conjugated histone h3k4me1 antibody in perm / block solution reserved in step 3.5 and add 10 l of this dilution to each well . note : use an h3k4me1 antibody conjugated using the r - phycoerythrin conjugation kit following the manufacturer s instructions . resuspend the cells in 200 l pbs+2% and transfer the samples to facs tubes for flow cytometry analysis . lymphocytes from a c57b/6 mouse were processed into a single cell suspension according to the protocol and counted using a standard hemocytometer . cells were seeded in triplicate at 2 x 10/ml in t - cell media in 15 ml conical tubes and left untreated , or stimulated with 1 mg / ml soluble anti - cd3 antibodies ( clone 4c11 ) for 3 hr at 37 c in a standard tissue culture incubator . dead cells were stained and then cells were then surface stained using fitc - cd8 and apc - cd4 antibodies according to the protocol . histone accessibility was analyzed via flow cytometry ( figure 4 ) . in both nave cd4 and cd8 t cells , the mean fluorescent intensity ( mfi ) is low , signifying a condensed chromatin state . as cells are activated with anti - cd3 antibodies the mfi increases significantly ( p < 0.001 , student t - test ) indicating that chromatin has decondensed . ( a ) the spleen is pressed against the frosted surfaces of two microscope slides . ( b ) the slides are moved back and forth against each other releasing lymphocytes into a 100 mm petri dish until the remains of the spleen are white . figure 2.using a syringe stopper to press remaining red pulp through a 70 mm cell strainer . ( a ) red pulp remaining after a spleen is processed into a single cell suspension and passed through the 70 mm cell strainer . ( b ) the stopper portion of a 3 ml syringe is used to gently press remaining red pulp through the cell strainer . ( c ) after using the syringe , there should be virtually no red pulp left in the cell strainer . ( a ) centrifugation of a single cell suspension generated from a single mouse spleen prior to ack lysis . ( b ) after ack lysis of red blood cells , the cell pellet should be white . ( b and c ) t cells were left untreated or activated with 1 mg / ml soluble anti - cd3 antibodies for 3 hr ( in triplicate ) . cells were then analyzed by flow cytometry to determine chromatin condensation in cd4 cells ( b ) and cd8 cells ( c ) . data are the means  standard deviation of the mean fluorescence intensity of h3k4me1 staining . * p < 0.001 ( student s t - test ) please click here to view a larger version of this figure . table 1 : troubleshooting guide . a quick reference to common issues and possible solutions . we developed a protocol that allows for the assessment of chromatin condensation in t cells . it relies on the simple observation that histone h3 antibodies can not access their epitopes readily in nave cells , but upon t cell activation , these same antibodies are able to bind to their epitopes . by comparing the mfi of histone h3 staining between treatment groups , the relative degree of condensation or decondensation can be determined . we have used this protocol to determine relative condensation status during thymocyte development and during t cell activation . we have also used this protocol to investigate the mechanisms that control the decondensation process . this protocol relies on the use of a histone h3 antibody to detect chromatin condensation status . since there is no global change in histone modification during t cell activation , we are able to use an h3k4me1 antibody to assess chromatin status in this protocol . we have used antibodies raised against unmodified histone h3 ; however , the signal produced was much weaker overall . in our experience , antibodies raised against modified histone h3 work better in immunofluorescence and flow cytometry assays , while antibodies against unmodified histone h3 work better in western blot . it must be noted that it is also possible to use antibodies raised against other histone proteins , although we have not attempted it . the perm / block and histone h3 antibody steps are the most critical steps in the protocol . the amount of perm / block solution used needs to be optimized each time the stock perm solution is made . a typical experiment involves comparing chromatin status in nave t cells to those activated for 3 hr ( figure 4 ) . one should choose the dilution that produces the greatest change in mfi over the time period analyzed . the stock solution can be diluted in pbs + 2% to decrease perm / block strength by first resuspending the cells in as much as 100 ml pbs + 2% after step 3.4 and then adding the perm / block in step 3.5 . a similar pilot experiment should be used to test different dilutions of the fluorescently conjugated histone h3 antibody each time a new batch of antibody is labelled . these steps are especially critical to successful detection of condensation differences early in t cell activation ( e.g. , within 3 hr of activation ) . occasionally , there are cells that do not get permeabilized and thus will not stain with the histone h3 antibody . this may happen if the cells are not resuspended well when adding the perm / block or if the perm / block is not strong enough . these cells will appear as events pressed against the axis when visualizing a histogram of histone h3 staining . since these events will skew the overall mfi , these events can be omitted from analysis by gating the normal distribution of histone h3 positive cells . the inclusion of a fixable dead cell stain allows for the assessment of cell viability in the assay . this is absolutely critical when manipulating t cell activation because certain stimuli can induce cell death . in such a case , the histone h3 antibody can bind histones in dead cells differently than live cells , leading to misinterpretation of the results . this protocol is designed for use in 96-well plate format , permitting a high throughput analysis of chromatin status . a spleen from a 6 - 8 week old female mouse since the staining protocol requires 2 million cells per sample , one can easily assay multiple treatment groups and time points in triplicate with a single spleen on a single 96-well plate . it is possible to perform the protocol with less cells per sample ; however , due to the number of centrifugation steps and the inherent loss of cells at each of these steps , it is not advisable to lower the number of cells by much . we used this protocol to examine chromatin status in cd4 t helper cells and cd8 cytotoxic t cells . the protocol could easily be adapted for the examination of chromatin in other lymphocyte subpopulations by using antibodies against population - specific surface markers . this protocol could also easily be adapted to other cell types so long as antibodies recognizing relevant surface markers are available and proper fixation / permeabilization conditions are known .