The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

The overall goal of this procedure is to assess the chromatin condensation status in T cells. This method can help answer key questions in the field of T cell activation. Specifically how chromatin controls clonal proliferation.

The main Advantage of this technique is that it can detect subtle changes observed during the earliest stages of T-cell activation. Following euthanasia of six to eight week old isogenic mice, spray the animals profusely with 70%ethanol solution and orient them so that the head is facing the left using forceps. Lift the skin upwards and away from the body.

Then cut a small notch through the skin near the abdomen. 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. Next, use forceps to lift the peritoneum and then make a small incision to expose the spleen. Remove the spleen with the forceps and use scissors to tease away any remaining adipose and connective tissue that is still attached to the spleen.

Place a spleen into a 50 milliliter conical tube containing 10 milliliters of PBS supplemented with 2%FBS hereafter referred to as 2%Leave the tube on ice until ready to continue with the procedure. When all of the spleens have been collected, decant them into a sterile 100 millimeter tissue culture dish. Obtain two frosted microscope slides and hold the slides so that the two rough frosted surfaces face inwards towards one another.

Then wet the slides in the 2%solution from the Petri dish. Place the spleen between the frosted surfaces of the slides and with the edges still submerged. Grind the spleen gently by moving the slides back and forth against one another.

Continue grinding until all cells have been released and the remains of the spleen appear white. Then draw up the cell suspension in a pipette and filter it slowly through a 70 micron cell strainer into a new sterile 50 milliliter conical tube. Add five milliliters of 2%PBS to gather the rest of the cells using the stopper portion of a three milliliter syringe.

Gently press the red pulp containing the remaining cells through the strainer. Make sure to press both the bottom and sides of the filter to ensure that all red pulp has passed through the filter. Next, rinse the tissue culture dish with 10 milliliters of 2%and then use this solution to wash the slides syringe, stopper, and dish to recover any remaining cells.

Pass the solution through the same 70 micron cell strainer and into the 50 milliliter conical tube. Then centrifuge the filtered cell suspension at 300 times G for five minutes at four degrees Celsius. Gently decant and discard the snat taking care not to disturb the pellet with a trace amount of 2%left in the tube.

Cap the tube and flick the pellet until the pellet is completely resuspended in the residual solution. Next, add two milliliters of act lysis buffer per spleen to the mixture. To lys the red blood cells, gently rotate and invert the tube for one minute to ensure that all of the cells come into contact with the lysis buffer.

Following the one minute incubation, bring the volume of the cell suspension to 50 milliliters by adding 2%to the tube. Invert the tube 10 times and then centrifuge the solution for five minutes at 300 times G and four degrees Celsius. Gently decant and discard the snat taking care not to disturb the white pellet.

Leave a trace amount of 2%in the tube, cap it and then flick the pellet to resus. Suspend it. Next, add 10 milliliters of T-cell media and further resus.

Suspend the pellet by gently pipetting up and down. Then filter the suspension through a new 70 micron filter into a fresh 50 milliliter conical tube. Use another five to 10 milliliters of T-cell media to rinse the original tube and pass this solution through the 70 micron filter as well.

Count the cells and access viability. Using the Trian blue method and a hemo cytometer, keep cells on ice until they are ready to be stained. Pellet the cells by centrifuging them for 10 minutes at 300 times G at four degrees Celsius.

Then resuspend the cells in T-cell media to a concentration of 10 million cells per milliliter. Transferred 2 million cells in 100 microliters of media to the wells of a U bottomed 96 well tissue culture plate centrifuge. The 96 well plate for 10 minutes at 300 times G and four degrees Celsius.

Once finished, remove the snat from each well by flicking the liquid within the wells into the sink and dabbing the plate on a clean paper towel. Next, resuspend the cells in 200 microliters of PBS to wash the cells, followed by centrifugation at 300 times G for 10 minutes at four degrees Celsius. Remove the SUP natant by flicking the plate again, and then add 100 microliters of freshly prepared fixable red dead cell stain to each.

Well incubate the plate at four degrees Celsius for 30 minutes while it is protected from light. Then centrifuge the plate for 10 minutes at 300 times G at four degrees Celsius. At this point forward, perform all work with the plate in the tissue culture hood with the light turned off.

Remove the supra natant by flicking the plate and then add 100 microliters of FC block solution to each.Well. Next, prepare anti CD four and anti CD eight primary antibodies by diluting them with PBS in accordance with the manufacturer's instructions. Add 100 microliters of the diluted antibodies to their respective wells and incubate the plate for 30 minutes at four degrees Celsius.

While it is protected from light following incubation of the surface stain, wash the cells twice in PBS After the last wash, add 100 microliters of freshly prepared 4%paraldehyde to each well and incubate the 96 well plate for five minutes at room temperature in the dark. Next, wash the cells twice in PBS and then add 40 microliters of a permeation blocking solution containing PBS with 2%FBS 0.02%Trix 102%normal rabbit serum into each sample. Well mix each well by gently pipetting up and down, taking care not to make bubbles.

Then incubate the plate at room temperature for 45 minutes in the dark. Next, add 10 microliters, a fluorescently conjugated histone H three K four ME one antibody diluted in the permeation blocking solution. To each, well mix the wells again by gently pipetting up and down The labeled histone.

H three antibody concentration may vary and must be titered each time it is made. Incubate the 96 well plate in the dark for one hour at four degrees Celsius. Lastly, wash the cells twice in 2%and then resuspend them in 200 microliters of 2%For fax analysis, histone H three antibodies cannot access their epitopes readily in naive cells, but upon t T-cell activation, these same antibodies are able to bind to their epitopes.

Here, the mean fluorescence intensity of histone H three staining is shown in CD four positive and CD eight positive cells that were either untreated or activated with one microgram per milliliter of soluble anti CD three antibodies for three hours. The significant increase following activation indicates that the chromatin has decon condensed in the treated samples Once mastered. This experiment can be done in 11 to 14 hours depending on the experimental design.

After watching this video, you should Have a good understanding of how to use flow cytometry to assess the chromatin condensation in T cells.