To FACS sort cells based on a nuclear antigen, I have used a fix/permeabilize/stain protocol (Transcription Factor Buffer Kit, BD Pharmingen) with success several times. I'm able to pellet the cells just fine during the staining steps (there is only one fixation step--it's before the staining). The staining works well, with low background, and the % positive cells counted by flow cytometry is as expected. However, even if I centrifuge at 10,000 g for 20 min, I cannot pellet these cells after the sort. There are over 1 million cells per tube, which should give a visible pellet. I tried Amicon Ultra centrifuge filters as an alternative way to separate the cells from the buffer, but this didn't work very well either.
The cells are HEK293, suspended in PBS+/+ before sort, and sorted into PBS in a 15mL Falcon conical tube. Any suggestions are greatly appreciated!
In my opinion, 10,000 x g for 20min its far too much force/speed for cells you are likely shearing / damaging the cell membranes and the cells are becoming lysed. Just my hypothesis.
Can you see cells in a microscope before centrifuge after sorting?
If not,
Did you add some medium (1-2 ml) into the collection tube before sorting? Sorted cells may stick on the wall of tube which can be very hard to centrifuge.
Thanks Zengli for your quick response. Yes, I can see cells in the microscope after sorting and before AND after centrifuging. I added some PBS to the tube into which the cells are sorted, but not medium.
Thank you Craig Silver for the response. I first tried centrifuging at 500g for 10 min, a typical speed for these cells...this did not work. Neither did 2000g for 15 min. The 10000g was out of desperation. Also, the cells are fixed/permeabilized so they can't really lyse....and I'm able to see them under the microscope after centrifugation.
Dear Anna,
I do not have experience with cell sorting, but I have analysed fixed and immuno-stained cells by flow cytometry.
Like you, I also noticed that cells washed with PBS are hard to pellet and seem to stick to the tube walls and tips. I believe it has something to do with PBS surface tension. Empirically, I solved the problem by adding 0.5% BSA to the PBS, which reduced surface tension and reduced cells sticking to tubes and tips as well. I would say 0.5% serum might work as well.
Good luck.
Thanks Jonas! I will give this a try.
Zengli, did the medium you added to the tube have serum?
Yes, I just used the full medium with 10% serum for cell culture.
Jonas is right, BSA should also work.
Additionally, I used swing bucket centrifuge so that any cells spin down will be at bottom of tube. I do not recommend centrifuge with fixed angle.
The pellet has landed! Thank you all for your help!
The cells pelleted by using PBS + 2% FBS in the FACS buffer, and spinning for the maximum speed in a swinging bucket rotor. Additionally, I got even more cells to pellet when I coated the sides of the tube into which they were sorted, by adding 1 mL of serum to the tube, capping it, and rotating it for a little while.
Cheers!
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