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

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

Transcript

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.

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