Cell counting of Strongly adherent cells
Posted 20 November 2002 - 08:32 AM
Thanks whoever replies to this topic.
Posted 21 November 2002 - 09:18 AM
You can try adding Trypsin and knock the side of your tissue culture plate to loosen the cells once the cells are detached from the bottom you can start resuspended the cell by pipetting up and down. Then wash the plate with your growth media (media which the cells are normally in) crucial that the growth media has FBS/FCS as that will inactivate trypsin. Spin down and resuspend in fresh media the spun down cells and take 10 ul of the resuspended and mix 90 ul of trypan blue (1:10 dilution). Take 10 ul of the trypan blue/cell mix and add to hemocytometer to count cells. If the cells still appear to be clump together repeat the trypsin addition and incubate a bit longer.
Posted 13 February 2003 - 12:36 PM
You might also try diluting the trypsin-EDTA in the same saline you use for rinsing the cells. I've gone up to 1:10 dilutions depending on the cells. It sounds counter-intuitive to use less trypsin - but you're trying to balance getting the cells off of the surface without chewing them to bits.
The advice about collecting, spinning down, and resuspending cells in media with serum is also good. I don't know the size cell pellet you're working with, but I wouldn't go any bigger than a 5-ml pipet, and 2-ml might be better.