DETERMINATION OF NITRIC-OXIDE GENERATION IN MAMMALIAN NEURONS USING DICHLOROFLUORESCIN DIACETATE AND FLOW-CYTOMETRY

A method for the rapid detection of intracellular nitric oxide (NO) ge neration in dissociated cerebellar granule cells using dichlorofluores cin (DCFH) and now cytometry was developed. DCFH can be oxidized speci fically by NO and this was assessed by 1) the use of SIN-1 (10 nM-100 mu M), an NO donor, that induced a concentration-dependent increase in dichlorofluorescein (DCF) fluorescence and 2) the use of hemoglobin ( 10 mu M), an NO-scavenger, that totally inhibited the increase of fluo rescence induced by SIN-1 (10 mu M). This assay was used to determine the ability of kainate to stimulate NO production in dissociated cereb ellar granule cells. Kainate (1 mu M-10 mM) induced an increase in DCF fluorescence that was partially reduced by N-G-nitro-L-arginine (1 nM -10 mu M), a nitric oxide synthase inhibitor (61.9% +/- 9.1), or hemog lobin (10 mu M) (55.0% +/- 4.1). The method described allows evaluatio n of the oxidation of DCFH to produce DCF as a parameter for measuring intracellular NO generation. The extent of DCFH oxidation by NO and R OS can be determined by using NO scavengers or NO synthase inhibitors. (C) 1997 Elsevier Science Inc.