Quantitation of lymphocyte intracellular free calcium signals using indo-1

The calcium-responsive fluorescent dye indo-1 has been used in lymphocyte suspensions to measure changes in internal free calcium concentration, [Ca 2+] i, in response to crosslinking of cell surface immunoglobulin. The quantitation of [Ca 2+] i requires that indo-am ester used to load the cells be completely hydrolyzed to the indo-1 form inside the cells. This is shown to be greatly facilitated in the lymphocyte by the detergent Pluronic F-127. The quantiation of [Ca 2+] i transients also requires an estimate of the fraction of the cells which contribute to the observed changes. The use of excessive amounts of intracellular dye can buffer [Ca 2+] i transients and this effect has been used to estimate the size of the pool of calcium which is available for release when the B cell is stimulated by anti-immunoglobulin.