SIMULTANEOUS FLOW CYTOMETRIC MEASUREMENTS OF CYTOPLASMIC CA++ AND MEMBRANE-POTENTIAL CHANGES UPON FMLP EXPOSURE AS HL-60 CELLS MATURE INTO GRANULOCYTES - USING [CA++]IN AS AN INDICATOR OF GRANULOCYTE MATURITY

Treatment of human leukemic HL-60 cells with N,N-dimethylformamide (DMF) induces them to mature until they reach granulocytoid morphology 3-6 d later. We have reported a maturation-dependent ability of these cells to respond to phorbol myristate acetate (PMA), as evaluated by membrane depolarization and by oxidative burst product formation (Newburger et al.: J. Biol. Chem. 259, 3771, 1984). More recently we have attempted to develop techniques for simultaneous evaluation of these parameters during HL-60 cell maturation. Here, we compare the cytoplasmic [Ca++] and membrane potential changes elicited by the chemotactic peptide fMLP via simultaneous measurement of individual cells in a fluorescence-activated cell sorter (FACS), as done previously for mature granulocytes (Lazzari et al.: J. Biol. Chem. 261, 9710, 1986). The stimulus-induced [Ca++](in) changes are detected with the fluorescent probe Indo-1 and reproducibly increase in magnitude for a subpopulation of cells as the cells mature into granulocytes. Ca+a+ responsiveness to formyl peptide is restricted to a subpopulation of HL-60 granulocytes which expresses receptors for chemotactic peptide and consistently increases in magnitude (in response to the same concentration of agonist) with maturation. In contrast, there is less consistency in the direction or magnitude of membrane potential changes elicited under the same circumstances from the same maturing HL-60 cells.