hi, i'm a neophyte at flow cytometry, so i'm hoping someone will be able to answer these questions. 1) i'm interested in monocytes and use CD14. Now, as i understand it CD14 stains monocytes brightly, and granulocytes dimly. So in all my samples i've been puzzled to find a large amount of bright CD14 cells in the region of a standard FSC-SSC plot that is the granulocyte region (higher SSC then the monocyte region). I still see that classic cluster of monocytes, but there are also these other cells. So we sorted these cells - hi SSC/bright CD14 - and looked at them under EM and there are plenty of neutrophils there, and monocytes. So whats the story with these cells? are there hi-SSC monocytes? why are these neutrophils bright for CD14? 2) This isn't really a flow question but maybe someone knows... We tried to isolate a rare population of CD71/CD14 double positive cells, but only got 8500 cells from 20 cc of blood. The EM people weren't able to prepare this little amount for EM. So, aside from using tremendous amounts of blood and antibody and flow time to get several hundred thousand of these cells, Does anyone know of any EM techniques for preparing small amounts of cells? thanks alot. --- Seth L. Ness M.D., Ph.D Fellow in Human Genetics Department of Human Genetics Mount Sinai School of Medicine Phone:212-241-6947 Fax:212-860-3316 sln8@columbia.edu Ness Gadol Haya Sham
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