Cell counting of Strongly adherent cells - (Nov/20/2002 )
Anybody has experience with Cell counting of Strongly adherent cells? The cell line I have is HCT116, which is a strongly adherent one. I tried many times but failed. I always find the cells gather tightly which makes my precise cell counting very difficult. Even pipetting up and down can't disperse them thoroughly.
Thanks whoever replies to this topic.
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.
You've probably tried this already, but just in case you haven't - make sure you're rinsing your cell layer with saline that doesn't contain calcium or magnesium, and trypsinize with trypsin-EDTA. Some of the clumping you're seeing may come from rough handling of the cells - believe me, I understand thumping on flasks to get the cells loose, but sometimes they don't respond well to it. They're already under attack from a protease, and sometimes the mechanical shear forces of rapping the flask are just too much. A cell that falls apart releases its DNA, which is apparently nature's perfect cell glue - and you'll get clumps.
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.
This is strange. for me HCT116 gets tripsinized in less than a minute. any way, to solve ur problem try to trypsinize the cells when it is 80% confluent. try to use non enzymatic cell dissociation agent along with the trypsin. u can get it from sigma. the purpose is to avoid clumping of cells. hope this will work.
Agree with senthil, always subculture prior to full confluence.
Also try washing x3 with PBS ( w/o Divalent cations), then wash x3 with VERSENE 0.05%( EDTA solution)