Quantitative characterization of crude synaptosomal 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 components, size, and intactness. Particle size ranged from a few tenths of a microm to greater than 4.5 microm. The viable dye calcein AM labeled 90% of the preparation, indicating that the majority of particles were intact and esterase-positive. 66% of the P-2 fraction is neuronal in origin, as demonstrated by labeling with an antibody directed against SNAP-2. An antibody directed against glial fibrillary acidic protein (GFAP) labeled 35% of the particles in this preparation. The mitochondrial dye nonyl acridine orange (NAO) stained 74% of particles, indicating intra- and extrasynaptosomal mitochondria. Gating analysis reveals that SNAP-25 is enriched in the larger particles. These results suggest that flow cytometry may be used to take advantage of the increased viability, yield, and convenience of the P-2 fraction for studies of nerve terminal function.
Gylys
K H
KH
UCLA School of Nursing, Los Angeles, California 90095-6919, USA. kgylys@sonnet.ucla.edu
Fein
J A
JA
Cole
G M
GM
D000818:Animals; D004396:Coloring Agents; D005434:Flow Cytometry; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009457:Neuroglia / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D012542:Scattering, Radiation; D013347:Subcellular Fractions / Q000737:chemistry; D013574:Synaptosomes / Q000737:chemistry*
D004396:Coloring Agents; D009419:Nerve Tissue Proteins