Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals

Begin with mice thymocyte cells in a multi-well plate. Add magnetic beads coated with anti-CD3 and anti-CD28 antibodies. To a subset of wells, add an appropriate concentration of the test kinase inhibitor. Incubate.

The antibodies bind to the cell-surface CD3 and CD28 receptors on the double-positive thymocytes and activate them, causing downregulation of T cell receptors, TCRs, and activation of cytosolic protein kinase. 

These protein kinases activate downstream signaling pathways, leading to increased cell-surface CD69 expression and cytosolic caspase-3 activation. Activated caspase-3 cleaves essential cellular proteins — initiating thymocyte apoptosis.

In cells treated with the kinase inhibitor, the inhibitors block the cytosolic kinases, modulating CD69 expression and caspase-3 activation.

Pipette in a fluorophore-tagged antibody cocktail that binds to the thymocytes' TCR, CD4, and CD8 co-receptors, and CD69 receptors. Treat the thymocytes with a buffer to fix and permeabilize them.

Incubate with a fluorophore-tagged anti-caspase-3 antibody that binds to cytosolic caspase-3. Analyze the thymocytes using flow cytometry.

Control double-positive thymocytes express the fluorescent cell-surface markers CD4, CD8, and CD69 and intracellular caspase-3 and show TCR downregulation. In contrast, inhibitor-treated thymocytes exhibit lower levels of fluorescent CD69 and caspase-3.

To begin the treatment of thymocytes with kinase inhibitors, use a multi-channel pipette to add 40 microliters per well of thymocytes to a small-volume plate. Put the plate on ice. Then, pipette one part of kinase inhibitors, DMSO, and dexamethasone, and four parts of the complete RPMI media into a 96-well plate.

Designate eight wells of the small-volume plate to untreated controls, four wells to dexamethasone-treated positive controls for cell death, by adding the diluted dexamethasone at the 5-micromolar concentration, and four wells to vehicle-treated controls by adding 0.5 microliters of the diluted DMSO. Next, from the corresponding wells of the inhibitor plate, add 0.5 microliters of each diluted inhibitor to the 96-well plate.

To begin thymocytes' stimulation, first, make sure anti-CD3 and CD28 beads are uniformly resuspended. Next, wash 1 milliliter of the beads with 2 milliliters of the PBS buffer. Put the tube on a magnetic stand to separate beads from supernatant, and aspirate the solution. Then, resuspend the beads in 1 milliliter of the complete RPMI medium.

Add 10 microliters per well of the bead suspension to each inhibitor-treated sample, the four DMSO-treated samples, and four of the 8 untreated samples. Add 10 microliters of the complete RPMI to the remaining four untreated wells.

Use a microplate orbital shaker to gently agitate the plate. Next, incubate the thymocytes in 37 degrees Celsius and 5% carbon dioxide environment for 17 to 20 hours or overnight.

Prime an automated laminar flow plate washer, first, with 150 milliliters of ethanol-Tween solution, then, with deionized water supplemented with 1% Tween-20, and finally, with FACS wash buffer. At the end of the incubation, using the plate washer system, wash the plate with FACS wash buffer using a nine times washing cycle. Each wash adds and removes 55 microliters of wash buffer by laminar flow, resulting in exponential dilution of the reagents in the wells.

To stain surface antigens, first, dilute one unit volume of anti-CD3, anti-CD4, anti-CD8, and anti-CD69 antibodies in 100 unit volumes of the FACS wash buffer. Then, resuspend the cells in 25 microliters of the staining antibody mixture. Use a microplate orbital shaker to gently agitate the plate, and then leave the plate on ice for 30 minutes.

To fix the cells, first, wash the plate with 55 microliters of FACS wash buffer using nine times washing cycle as described before. Then, add 50 microliters of the fixation and permeabilization buffer to each well. Mix well, and incubate the plate on ice for 30 minutes. After the incubation, prepare a 1x perm/wash buffer by diluting 25 milliliters of the provided 10x stock in 225 milliliters of ultra-pure water.

Prime the plate washer with 1x buffer, and wash the plate with 55 microliters of the 1x buffer using a nine times washing cycle as described before. Mix 1 milliliter of the anti-caspase-3 antibody with 2 milliliters of the 1x perm and wash buffer. Add 25 microliter per well of the mixture to the fixed cells.

Use a microplate orbital shaker to gently agitate the plate. Leave the plate on ice for an hour. At the end of incubation, repeat the wash step, nine times with the 1x perm and wash buffer. Then, add 25 microliters of the FACS wash buffer to all wells of the plate, pipette up and down a few times to mix the solution.

Finally, transfer the mixed samples into microtiter tubes. Top up the tubes with the FACS wash buffer to a total volume of 200 microliters and proceed to the flow cytometry analysis.