A microplate assay for the detection of oxidative products using 2′,7′-dichlorofluorescin-diacetate

A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2′, 7′-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucyl-phenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37°C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 × 10 5 − 1 × 10 6 cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples ( r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates.