2 replies to this topic

[cmccray]

I have been growing these TRL-2 knockout microglia (mouse cells) for 18 days now. They are suppose to be adherent yet ~99% are still floating in suspension. I'm using DMEM w/ 2mM L-glut, 1mM NaPyr and 10% FBS w 5% CO2, I was following a similar cellline protocol for basic growth parameters but it said the cells should take 5-7 days to attach. I seeded into a T-75 flask. I was using non-coated flasks and decided to exp. w coated flasks. I have tried collagen 1, Lysine, and Lamminin treated flasks in T-25s which normally the volume of media would be about 5mL. I even tried setting them up in 2mL for 72 hrs in hopes that the tiny cells would find their way to the bottom, then added the other 3mL of media. All the floaters are dying off now or are dead.

Still haven't seen any improvement. When I set up p.0, I had a viability of 35%. Now my parent T-75 is showing some cells finally adhering in small colonies (Only up to maybe 5% confluency) and they look super healthy but the rest are just floating and dying. I have done media replacements and replenishments regularly. This vial originally came from a depositor and I can't get another one. Still no such luck. I'm out of ideas.


Any suggestions would be greatly appreciated!

[bob1]

Have you tried seeding in a smaller volume?  Often brain cells have very specific limits for density - too high or too low and they won't attach or grow.

[swirskilab]

Hi, were you able to resolve your problem? I'm having similar issues with an ATCC line (EOC 13.31), grown under the conditions ATCC suggests and in tissue culture treated flasks. The cells remain round, even when they adhere.