Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye

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.