Wash cells or not after trypsinization? - (May/03/2005 )
I agree with kknb.
I avoid centrifugation as much as possible, because this step can sometimes be no less harmful to cells than trypsinization.
After adding trypsin, I immediately aspirate trypsin and incubate the petri dish at 37C until the cells come off (the incubation time depends on the kind of cells).
I then suspend the cells in media by pipetting up and down. This method minimizes the influence of tryosin and generally works well unless the cells are attached to the dish very firmly.
assuming trypsin is inactivated by addition of the media the fact you resuspend cells in media before spining is good. Moreover as you discard the supernatant after centrifugation , the amount of trypsin that remains in the pellet is very low. Finally as you resuspend your cells in fresh media, inactivation occurs again (if there was active trypsin remaining)...
You can also directly plate the cells after suspending them in media without spinning.
the only advantage of washing cells after trypsinization - if your cells are very "sticky" and tend to form clumps, but extra centrifugation step will kill some of the cells
I completely agree with biosky.
I always remove the trypsin after ensuring that the cells have detached; then add the medium to aspirate & re-plate. This way you don't have to worry about the amount of trypsin in the newly-plate cells.
I always remove trypsin too. Depending on the type of cells, I separate them by quick spin or I just let Trypsin drain on the bottom of the flask for 10 min in the incubator.
Hardly anyone washes their cells after trypsinization!! I pass 15-20 flasks at a time everyday...imagine the time wasted in washing each and every flask after trypsinizing!!
The answer is : No, you dont have to wash your cells after you trysinize them. I have never done it for anykind of cells , and it ALWAYS works!!
I have used both ways of tyrpsinizing my cells. I only add ~2mL of 1x Tryspin EDTA and spin my cells at 1k rpm for 5min, aspirate supernatant with a pasteur pipette and then resuspend cells in media...they seem fine after that. I have tried deactivating trypsin and then spinning and still got the same results...It works either way...
I try to minimise the time my cells are exposed to trypsin as they don't seem to give the same responses if I'm a bit slack. But I find neutralising the trypsin with media works fine - I've never washed my cells.
i was trying to separate out the cells after trypsinisation. But unfortunately evenafter 2000 rpm 10 mnts.. cells were still in the suspensiion (clump).
could anyone help me to solve this issue. ( I used 10 ml of trypsine/versine for 75 cm2 flask) soln and incuated for 10 mnts