QUANTITATIVE MULTIWELL MYELOID DIFFERENTIATION ASSAY USING DICHLORODIHYDROFLUORESCEIN DIACETATE (H2DCF-DA) OR DIHYDRORHODAMINE-123 (H(2)R123)

It is well established that the fluorescent probes dihydrodichlorofluorescein diacetate(H2DCF-DA) and dihydro rhodamine 123 (H(2)R123) can be used to detect the respiratory burst response of mature myeloid cells. We describe a simple, fast and quantitative assay for myeloid differentiation based on the oxidation of these probes, which can be performed from start to finish in 96-well dishes. A bis(acetoxymethyl) ester of H2DCF-DA, 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CODCF-DA) is also capable of detecting the respiratory burst, but is less suitable than H2DCF-DA or H(2)R123 in our system. The amount of fluorescence produced can be quantified using a calibration curve, and values can be normalised to cell numbers using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylte-trazolium bromide (MTT) cell proliferation assay. Results are expressed as 'equivalents of soluble fluorescein' (ESF) produced per cell under the defined reaction conditions. The extent to which HL60 cells reduce MTT is unaffected by differentiation induced by retinoic acid or 1 alpha,25-dihydroxyvitamin D-3, and normalisation of fluorescence values using the MTT assay appears to be valid for a wide range of myeloid cell lines and differentiation inducers or cytokines.