Cells sensitive after replacing media - what am I doing wrong? - (Jul/04/2011 )

I'm working with FlpIn 293 cells and trying to run a DMSO sensitivity assay on a 12-well plate.

I plate cells to about 25% and wait until 70~80% confluency before I replenish the media and add DMSO. Problem is, the cells are detaching and/or become terribly sensitive after I replenish the media.

I'm using 1ml of media for each well, so I take 1ml pipette to remove old & add fresh media for each well one by one.

I suspected decreasing temperature might be the culprit, so I've done everything to minimize my time at the bench and to make sure the media is at the right temperature, but the entire cell population in general are in much poorer shape after media replacement. Is there something I'm missing that could be causing this?\


TIA

Are you adding a selective agent to keep the FLP-in plasmids in the cells?

Aren't 293 cells very sensitive to pressure? I don't think they are as strongly adherent as many other cells. I have had cells detaching from plates if I pipette too hard directly onto them, so I usually put the media into the wells gently down the side of the well rather than directly onto the cells, which generally solves the detachment problems. I don't know how you do it currently but just a thought.

Thanks for the replies!

bob1 on Wed Jul 6 02:57:07 2011 said:

Are you talking about transfection step? I am using selective agent (hygromycin B ) but 'm working with stably integrated cells.

philman on Fri Jul 8 11:29:53 2011 said:

You're right they are very sensitive to pressure (I found out the hard way)...I actually thought this was the problem first time around because, like you said, I was pipetting right onto the cells and you could see the detachment coming mostly from the spot right below where the pipette was at. But since then I've gone through couple more cultures and while I was able to minimize detachment from pipetting pressure, I still notice that majority of cells are in a very poor shape...



It looks like this is an inherent weakness of 293 cells and I'll just have to give myself more backup in case of failures...

Stable integrants can still loose the integration through methylation; you need to keep some minimal selective pressure on the cell line to ensure that the site does not get lost.

The reason I asked was to check whether the cells were under some selective pressure but might have lost the integration.