A DOUBLE-LABELING FLUORESCENT ASSAY FOR CONCOMITANT MEASUREMENTS OF [CA2+](I) AND O-2 PRODUCTION IN HUMAN MACROPHAGES

To measure intracellular free Ca2+ concentration ([Ca2+](i)) and super oxide (O-2(.)) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O-2(.)-sensitive dye dihydrorhodamine-123, which becomes fluorescent in its oxidized form, rhodamine-123. We describe a new double-dye technique whereby tile kinetics of both [Ca2+](i) levels and O-2(.) production can be monitored simultaneously. This technique was developed in the dimethylsulfoxide-differentiated monocytic-like U-937 cell line (not equal U-937), validated by comparison with single dye measurements and applied to human alveolar macrophages. The chemotactic peptide N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine induced in both cell types a similar transient elevation in [Ca2+](i), followed within seconds by a sustained increase in O-2(.) production, which was however 4-fold weaker in not equal U-937 cells. These results indicate that O-2(.) production is an early event following the stimulation of human alveolar macrophages. This new double-dye technique may be relevant to other O-2(.) ion-producing cells and could help to define more precisely the kinetics of the events leading to this biological response.