Im new in cell culture, I really stuck and dont know what should i do? Ive done seeding 96 well plate but after crystal violet, there is no cells or the number of cells is terribly low. my result in 570 nm is not valuable. Ive done three time and all nothing. before crystall violet the cells was seen in microscope and there is cells in wells
Are you cells adherent or suspension? Wat is the number of cells you have started with? Do you wash the cells before or after treatment?
I usually seed cells in the range of 20000 to 40000 per well. Washes are to be done very carefully.
For Adherent cells - could be loosely bound to surface (Use corning cellbind plates). Washes should be done carefully.
For suspension cells - the cells need to be settled down by centrifuging the plate to remove the media and then add wash solution and repeat the centrifuge.
Hope this answers ur problem
is there any chance your crystal violet is not properly made which lysis your cells?
You should answer Dr. Ganji's qustions to better troubleshoot. But also you should never stain cells with an toxic antiseptic before you seed. If the cells are gram positive, negative or mammalian, there will be inhibitory effects on the growth when you prestain with crystal violet. An easy test to verify, just seed directly without staining first, that will probably solve your problem.
adherent cells is seeded and the number of cells should be 5000 per well in 96 well-plate....i wahsed my cells before trypsinizing only
As suggsested by Dr. Xiong, 5000 adherent cells can not vanish, if not being lysed by the solution you are using.
- Badges
- Science method