are cells prone to swelling in PBS after sorting - (Jul/21/2009 )

I noticed quite some times that the size of the cell pellet I collected after sorting got shrank (significantly) after I washed the cells with PBS. Does anybody else has the same issue? Is there any way to avoid the loss of cells? Thanks a bunch!

The cells I am collecting are really precious and it is painful to see that happening.

TracyDuke on Jul 21 2009, 09:46 AM said:

Cells should not shrink in PBS, so you may want to use another lot of PBS, just in case if has gotten concentrated after evaporation.

Other reasons:

You sure that it is not due to reduced number of cells after sorting?

Is your centrifugation speed right?

I would just count the number of cells before and after washing, and get a look at their size too.

Thank you Cellcounter!
I tried comparing by counting the cells before and after washing. And I found that the number of the cells dropped to about 1/5!! I was shocked. Is it possible that because the cells go through some sort of presure from the FACS machine which makes the cells fragile and therefore easily swell/burst in PBS?

TracyDuke on Jul 25 2009, 12:20 AM said:

Probably not. Check the supernatant after centrifugation, I think you may find your "lost" cells there.

Anyway, cells do not like being in PBS (or any other protein-free solution) for too long. Do you sort cells into tubes containing some medium/protein? I find that tissue culture medium (bicarbonate-free, pHed to 7.2) containing 5% FCS is optimal for cell collection and washing. If you need serum-free, medium plus 0.1% BSA should be fine.

You need bicarbonate-free medium so you don´t get pH changes in the absence of CO2 gassing.

Hope this helps.