Hi,

We're trying to do FACS sorting on cells which grow in suspension (specifically, DG44, which is a CHO cell line which grows in suspenstion).
We're having problems maintaining their viability after sorting.

Cells are sorted into 50% fresh medium:50% conditioned medium.
Sorting is done in an isotonic solution (not PBS), at r.t.
Should we try changing to PBS? do it at 4C?
should cetrifugation of the cells after the sorting (to get rid of isotone) be avoided?
Or is it all just a question of the pressure on the cells during sorting.
note: when we check viability directly after sorting (with propidium iodide) the cells look fine.
but they don't survive to the next day.
Any suggestions?

Any ideas? Anybody?

This is an interesting problem. I've done a fair amount of single cell sorting using a CHO cell line and have had some difficulties. Sometimes I get lots of clones sometimes very few. Partly, I think it may simply be the machine set-up for getting the cells into their single wells (not your problem as I understand it), the other possibility is that the cells are not in an optimal state before sorting. Are your cells overgrown? That may add to their stress.

We generally sort in PBS, although I had one colleague who would sort with another type of solution containing glucose to aid viablity, but I'm sorry I don't know the reference. If your suspicious of the isotone, then try switching it?

Although I'm not familiar with DG44, most CHO cell lines should be tough. Sorting them at 4°C is preferable, but I wouldn't say absolutely needed. Your not sorting for hours on end are you?

Perhaps the conditioned media is a problem if it comes from overgrown cells? Cells make growth inhibitors as well as stimulators. Maybe you could just try fresh medium.

Unless the number of cells you sort are few, in which case you can sort them straight into culture medium, I generally spin them down to get rid of the sort buffer etc. This all depends on how many cells your sorting.

Are you sorting on a newer high speed machine? If yes, maybe you want to turn it down from '11' (Spinal Tap movie joke-if you don't get it).

Sometimes these problems are mutli-parameter and it looks like you have come up with a good list of potential problem areas. It may actually be a combination of things. Overgrown cells stressed by sorting and then tossed in conditioned medium with some growth inhibitor could be killing them. Hard to know. Hope this helps??

Thanks.
We checked and it's probably the isotone solution.