IMPROVED SENSITIVITY IN FLOW CYTOMETRIC INTRACELLULAR IONIZED CALCIUMMEASUREMENT USING FLUO-3 FURA RED FLUORESCENCE RATIOS

Measurement of changes in intracellular ionized calcium concentrations ([Ca2+](i)) has proved to be of wide use in the study of cellular res ponses to activating stimuli. The fluorescent dye Indo-1 has successfu lly been used in flow cytometry for this purpose, and when used as a r atiometric indicator it provides optimum sensitivity and accuracy. Unf ortunately, this dye requires ultraviolet (UV) excitation which is oft en not available. We show here that similar results can be obtained us ing a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser Line. With appropriate concentrations of the two dyes, the magnitude of response with the fl uo-3/Fura Red ratio is greater than that achieved with indo-1, while t he intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescen t detection of PE-labeled antibodies to enable [Ca2+](i) measurement w ithin cell subsets. (C) 1994 Wiley-Liss, Inc.