I need help in detaching monocytes from my plate. I'm seeding PBMCs for overnight culture (adding LPS) in a 96 well plate (polystyrene), stain these cells the next day and use FACS to analyse monocytes in detail. However, I rarely see CD14+ CD16+ cells. I assume they get lost because of adherence. Do you have any protocol for detaching the monocytes after incubation? I have read about PBS + EDTA and Trypsin so far. Thanks in advance!
You can just use a Cell Scrapers, and gently harvest cells from the plate or flask..
Dear @nils, first of all if you want your cell to be in suspension you must use shaker in incubator at low speed which will not allow monocytes to settle on surface.
Or you may add high concentration (20% or so) of FBS, making your medium viscose, slowing the settlement of cells.
Or you may use trypsin EDTA or Accutase for dissociation with harsh pipetting.
Cells scraper would not be successful in 96 well plate.
But both the later method cause loss of surface markers.
Há um reagente chamado Tryple livre de vermelho de fenol, muito utilizado em cultivo de células mesenquimais, pode ser uma opção para remoção das células sem agressão mecânica e danos nas proteínas de membrana. Já usei para fazer caracterização celular por citometria e funcionou. Espero que consiga resolver. Boa sorte!!!
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