Hello, I am using now suspended cells called Jurkat T cells and I need to fix them with PFA. The problem that I found is that when I centrifugate after the incubation with PFA I lost most of the cells that I had, even if I increase centrifugation time (10 minutes) and increase the G (800 G).
Any suggestions? I have never used suspension cells and I do not know much about them.
Hi Maria,
use more G can help, go for 5' 3000g. You can even go further up, the fixed cells wont damage too much. Cells may also attach to the tubes wall.
Best
Christian
I've never done this, but in the past when our lab has encountered "sticky" substances we have used siliconized pipette tips/tubes. You might consider getting a sample of each to see if this helps recovery if the centrifugation increase doesn't solve your problem.
Fixed lymphocytes tend to adhere in a strip down the outer edge of microtubes during centrifugation instead of pelleting, which you are seeing. The suggestions above are very good. Also- you can try fixing the cells as a pellet, or suspending in a very small volume and keep the washes small to keep the cells in the bottom of the tube. A swinging bucket centrifuge instead of fixed angle would also help direct them towards the bottom of the tube.
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