Encyclopedia of Experiments: Immunology
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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.
