Staining cells for FACS at 4 degrees or ambient temperature

Question

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

• Strain

• Sort

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

Answer 1

You have several questions, please try to limit your posts to one question each. That being said, I'll try to address each of your concerns.

Does the cold cause these cells to apoptose?

No. The purpose of staining at 4°C and washing with cold buffer is twofold - to maintain viability (all cellular processes slow down at lower temps) and to prevent receptor internalization. This second point is especially critical as you're using a primary and conjugated secondary, which I really wouldn't recommend for sorting unless absolutely necessary, for example if the antibody you're using isn't available in a directly-conjugated format.

One thing I can do to minimize cell death is do a live/dead stain (we just added that capability).

Live/dead stains are generally a good idea, but make sure the stain you're using isn't cytotoxic. L/D should always be the last staining step before running your samples. A better way (if your sorter supports it) is to use forward scatter (FSC) and side scatter (SSC) height and area measurements to discriminate single, appropriately-sized cells, then gate on them first before gating on your antibody stain (FITC or PE or whatever your secondary is).

The other thing I read was to use 50% FBS in your collection tube.

I'm not sure how much that will help, especially 293s, which are pretty tough. Just make sure you're diluting the collected cells enough in medium to bring the serum percentage back down to 10%.

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?

As long as your samples are kept in the dark on ice or at 4°C, they should be fine for several hours at least (I wouldn't go overnight, though). Also, make sure your collection medium is chilled, as it can shock the cells to go from 4° to 37° too rapidly. Collect your sample, plate it, then put it at room temp or in the incubator (depending on your workflow) and let it warm up that way.

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?

Nah, don't worry about it. You can wait if you want, or add it right away - I would lean towards adding it more quickly than not, so you don't give unwanted cells any chance of growing out.