Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) partially inhibits rapid intracellular free calcium transients triggered by anti-immunoglobulin in murine lymphocytes.

The highly fluorescent probes Indo-1 and Fura-2 were employed to detect intracellular calcium responses in murine splenic lymphocytes following cross-linking of cell surface Ig. Inhibition by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) was rapid and showed a strong preference for the very transient phase of the response which has been identified as a mobilization of intracellular calcium. 12-O-Tetradecanoylphorbol-13-acetate had significantly less effect on the longer lasting increase in intracellular free calcium which involved an influx of extracellular calcium. Whole spectra were used as a check on transients, which were monitored at a single wavelength, in order to eliminate changes that were not calcium-dependent. It was found that such changes could arise from the association of Indo-1, or its acetoxymethyl ester, with phospholipid bilayers since this affected their fluorescence spectra. In addition, the loading of resting cells with dye esters was shown to be greatly enhanced by the inclusion of a small amount of the detergent Pluronic F-127 in the incubation medium. A spectral analysis of labeled cells showed that the extent of hydrolysis of intracellular dye was improved as well as the rate of uptake by cells.