Hi Something key in separating monocytes by FACS is is include some EDTA in your buffer the monoctes are suspended in, 5mM should do. Without EDTA monocytes disappear from the suspension, sticking to tubing, each other or something. You should be able to get 1 million monocytes from 20 ml blood from most individuals. So just ficol your cells, stain with CD14 (purity isn't that bad if you just use scatter) and suspend in PBS with 5mM EDTA and sort Simon Monard Aaron Diamond Center NYC
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