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 . 
 during a proper immune response , quiescent t cells become activated upon antigen presentation to their antigen - specific t cell receptor . 
 this leads to clonal proliferation of only those t cells that bear a receptor that recognizes the antigen . 
 chromatin decondensation is a hallmark of t cell activation and is required for t cells to acquire the ability to proliferate after antigen engagement . 
 this change in chromatin condensation can be detected using antibodies raised against histone proteins . 
 these antibodies can not bind to their epitopes in nave t cells as well as they can in activated t cells . 
 we describe how to simultaneously stain t cell - specific surface markers , track viability with a fixable dead cell stain , and measure chromatin status via intracellular staining of histone h3 proteins . 
 stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity ( mfi ) of the histone h3 stain . 
 chromatin decondensation during t cell activation is demonstrated as an increase in the mfi 
Introduction
Protocol
1. Single Cell Suspension of Lymphocytes from Mouse Spleens
2. Viability and Surface Staining
3. Intracellular Staining for Histone H3
Representative Results
Discussion
Disclosures