Encyclopedia of Experiments: Immunology
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Transcript
Begin with a mouse T cell suspension and introduce a calcium indicator dye — Indo-1-acetoxymethyl ester.
The dye enters the cells, where intracellular esterases cleave the acetoxymethyl group to form membrane-impermeable Indo-1, which exhibits distinct fluorescence in calcium-free and calcium-bound forms.
Add Fc receptor antibodies to block nonspecific interaction sites.
Introduce fluorophore-labeled antibodies to stain T cell subsets.
Remove unbound antibodies and stain with a viability dye that differentially stains live and dead cells.
In flow cytometry, initially detect dim fluorescence of live cells and identify fluorophore-tagged T cells, followed by measuring Indo-1 fluorescence within these cells.
Treat with anti-CD3 antibodies, which interact with the T cell receptor CD3 complex and activate T cell signaling pathways, inducing calcium influx into the cytosol.
Indo-1 dye interacts with the released calcium ions, shifting the dye's fluorescence from blue to violet.
The shift in Indo-1 fluorescence from calcium-free to calcium-bound states confirms T cell receptor-induced calcium influx.
