A double-labelling fluorescent assay for concomitant measurements of [Ca2+]i and O2. production in human macrophages.
To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye dihydrorhodamine-123, which becomes fluorescent in its oxidized form, rhodamine-123. We describe a new double-dye technique whereby the kinetics of both [Ca2+]i levels and O2. production can be monitored simultaneously. This technique was developed in the dimethylsulfoxide-differentiated monocytic-like U-937 cell line (not equal to 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 O2 production, which was however 4-fold weaker in not equal to U-937 cells. These results indicate that O2 production is an early event following the stimulation of human alveolar macrophages. This new double-dye technique may be relevant to other O2 ion-producing cells and could help to define more precisely the kinetics of the events leading to this biological response.
Bueb
J L
JL
Centre de Recherche Public de la Santé, Luxembourg.
Gallois
A
A
Schneider
J C
JC
Parini
J P
JP
Tschirhart
E
E
D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D008264:Macrophages / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D020112:Rhodamine 123; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism*
D012235:Rhodamines; D013481:Superoxides; D020112:Rhodamine 123; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium; D016257:Fura-2; D004121:Dimethyl Sulfoxide