I'm sorting a 293 derived line. One thing that is worrisome for me is cell viability after the sort. Usually I have been staining and washing at room temperature (on the benchtop) on a nutator. I have been reading that most sorting procedures indicate that you should be doing this at four degrees in the dark. It is my understanding that this helps with clumping, but is this at the expense of viability? Does the cold cause these cells to apoptose?
Currently my procedure is the following -
Using HBSS + 1 mM EDTA + 1% BSA (sort buffer) I suspend approx 10-15 million cells per/mL.
Stain with primary
Spin at 300 x g for 5 minutes
Add 15mL of sort buffer, spin again
Stain with secondary in 1 mL of sort buffer
Wash x2 with 15mL
One thing I can do to minimize cell death is do a live/dead stain (we just added that capability). The other thing I read was to use 50% FBS in your collection tube.
The other thing that might be of concern is that if we are doing many sorts, then some of the tubes may be sitting in sorting buffer for a long time in the queue. How long is too long?
Lastly, we use puromycin and blasticidin as selection markers for our libraries and we usually add these right to the sort after we replate. Should we wait a while?
Thanks for the advice