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

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 dihydro rhodamine-123, which becomes fluorescent in its oxidized form, rhodami ne-123. We describe a new double-dye technique whereby tile kinetics o f both [Ca2+](i) levels and O-2(.) production can be monitored simulta neously. This technique was developed in the dimethylsulfoxide-differe ntiated 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 cell s. These results indicate that O-2(.) production is an early event fol lowing 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.