Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T lymphocytes with fluo-3, a new fluorescent calcium indicator.
Cytoplasmic free calcium [( Ca2+]i) is a key intracellular messenger in many cell types. We have used fluo-3, a recently developed calcium probe, to study [Ca2+]i in resting and stimulated human peripheral blood T lymphocytes. The spectral properties of fluo-3 permit analysis of [Ca2+]i in flow cytometers with a 488 nm argon laser excitation source and fluorescein filter settings. The data obtained with fluo-3 are both qualitatively and quantitatively in good agreement with the data in the literature. After stimulation of T lymphocytes with the mitogens phytohaemagglutinin, concanavalin A and with OKT3, and anti-CD3 monoclonal antibody, a biphasic [Ca2+]i response was observed, with an early EGTA-insensitive [Ca2+]i rise, followed by an EGTA-sensitive sustained [Ca2+]i plateau. Non-mitogenic monoclonal antibodies directed against the CD5, CD28 and CD7 T cell surface antigens elicited [Ca2+]i rises only when crosslinked on the cell surface with goat anti-mouse IgG. Conversion of fluorescence data into absolute [Ca2+]i values by means of a non-disruptive calibration procedure, yielded a [Ca2+]i of 107 +/- 18 nM (mean +/- SD, n = 13) in resting T lymphocytes. Time-dependent loss of cellular dye content limits the precision of the calibration procedure in experiments of longer duration. We conclude that fluo-3 promisingly extends the potential application field of flow cytometers with 488 nm argon lasers to [Ca2+]i studies in T lymphocytes.
Vandenberghe
P A
PA
Department of Medicine, University of Louvain, Belgium.
Ceuppens
J L
JL
D000814:Aniline Compounds; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D002118:Calcium / Q000032:analysis*; D002138:Calibration; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000032:analysis*; D014966:Xanthenes
D000814:Aniline Compounds; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D014966:Xanthenes; C059715:Fluo-3; D004533:Egtazic Acid; D002118:Calcium