Washing n2a cells out of 75 cm flask? - (May/07/2012 )

Hello, all.

I've been considering as of late that I am using too much trypsin-EDTA to wash the n2a cells out of my flasks.

Here is how i've done cell washes for splitting as of late:
1) Use sterile, cold PBS solution and use this to initially wash cells with the hope of affecting fetal bovine serum residue so that...
2) Trypsin-EDTA can be used in 1 mL quantities in each 75 cm flask.

Generally, I swirl the trypsin-EDTA, wait about two minutes, swirl again, get impatient, pull out a long 1 mL pipette, start sucking up liquid and washing, and after a while, it starts to wash the cells off into a collective pool of cells in the flask corner as I tilt the flask.

Is there a better way?

I've read that after the PBS wash, to add the trypsin-EDTA, place the flask back into the incubator for about 5 minutes, and then attempt to wash cells off with the 1 mL long pipette.

My group has workewd with N2a cells for many years. We do not used trypsin to detach them as they do not tend to adhere very strongly on tissue culture plastic surfaces.

Once they reach 60-80% confluence, they can be detached by blowing jets of growth medium across the monolayer with a sterile Pasteur pipette or equivalent. We then centrifuge at 300 x g for 5 min and resuspend the pellet by Paseur pipette with minimal clumping.