Cell culture advice
So I work for a large research facility which means we use a lot of veroe6 cells to make lots of 12 well plates for assays. The past few months we have had issues with the monolayers being uneven in the wells and inconsistent confluency. We recently changed our processing to see if it fixes the problem and it hasn't. We have tried rocking each plate immediately after seeding, warming the plates, different well volumes, letting the plates sit before incubating. Nothing seems to help. We have had these issues across multiple lines (we have our own cryopreserved banks). All lines have tested negative for mycoplasma and endotoxin. Any ideas or advice? I'm putting our current process below.
For a t300
Wash twice with 20ml hbss
4 ml of trypsin incubator at 37 c for 4-6 min
Smack flask once
Nuetralize with 16 ml of media (keep the flask flat as this is done)
Mix flask 20 times
Seed new flasks at 8e6 for 3 day split
No feeds and we usually get counts between 1.2e6 and 1.8 e6