Cells Detach With Serum Free Media - (Mar/23/2006 )

Hi! I've looked through the posts and I know this has been discussed, but I just wanted some more input about this.

I'm using Hep G2s for MTT assays with polymers. After seeding in 96 well plates (complete media) I wait for a day or overnight before I aspirate and then add the polymer solution for 4 hours. When I wash with PBS or DMEM, I've noticed that I lose all my cells. If I wash with complete media, I don't lose as many. Has anyone experienced this?

What's odd is that my labmate incubates in serum free media, and he doesn't see any cell loss. Is there something that I'm missing? Do I have to wait longer for cell attachment? Should I coat my plates with collagen or something?

Thanks!

hi,

i have experimented the reverse effect on my lymphoma cells that is under starvation all of the cells attached firmly on the culture plate...

Seb

hi, first your PBS should be with calcium and magnesium.
second maybe your washings are vigorous? it is enough to wash once or twice not more.
and the best advise from me is to change the MTT to alamar blue....

cells usually detach when you supplement with hypotonic solution or hypertonic solution. try using different concentrations of the polymer and the washing solution
proviously i experienced the same problem with J774 macrophage cell line . but the problem got solved when i changed the washing buffer

Is your complete media WARM and everything else cold or AT by any chance?

cold cells go "brr" and wrap their arms around themselves, which means they can't hang on.

Okay, so that's NOT how it works, but the visual is a good way to remember.

Well I just tried some trial runs of different media, all either at 37 or room temp. and the cells didn't detach too much with complete media. They detached a little more with opti-mem, and the most with pbs. I even incubated them in the corresponding media for 2 hours or so and I still got detachment with washing.