Improved sensitivity in flow cytometric intracellular ionized calcium measurement 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 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.
Novak
E J
EJ
Department of Pathology, University of Washington, Seattle.
Rabinovitch
P S
PS
D000814:Aniline Compounds; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D006801:Humans; D007093:Imidazoles; D066298:In Vitro Techniques; D007211:Indoles; D007424:Intracellular Fluid / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D012680:Sensitivity and Specificity; D014966:Xanthenes
D000814:Aniline Compounds; D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; D007211:Indoles; D014966:Xanthenes; C082877:fura red; C059715:Fluo-3; C048960:indo-1; D002118:Calcium