Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo‐3/Fura Red fluorescence ratios

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Abstract
Measurment of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo‐1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using 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 fluo‐3/Fura Red ratio is greater than that achieved with indo‐1, while the intercellular variation in measurement is similar to that seen with indo‐1. Analyses can be simultaneously combined with immunofluorescent detection of PE‐labeled antibodies to enable [Ca2+]i measurement within cell subsets.