Quantitative characterization of crude spnaptosomal fraction (P-2) components by flow cytometry

Flow cytometry, which definitively identifies each particle as positive or negative with respect to fluorescent markers, is used to characterize the P -2 fraction (crude synaptosomal fraction) with respect to primary component s, size, and intactness. Particle size ranged from a few tenths of a mu m t o greater than 4.5 mu m The viable dye calcein AM labeled 90% of the prepar ation, indicating that the majority of particles were intact and esterase-p ositive. 66% of the P-2 fraction is neuronal in origin, as demonstrated by labeling with an antibody directed against SNAP-2. An antibody directed aga inst glial fibrillary acidic protein (GFAP) labeled 35% of the particles in this preparation. The mitochondrial dye nonyl acridine orange (NAO) staine d 74% of particles, indicating intra-and extrasynaptosomal mitochondria. Ga ting analysis reveals that SNAP-25 is enriched in the larger particles. The se results suggest that flow cytometry may be used to take advantage of the increased viability, yield, and convenience of the P-2 fraction for studie s of nerve terminal function. (C) 2000 Wiley-Liss, Inc.