I am currently working on in-vitro assay standardization where I have to centrifuge cells to wash cells after Flow Cytometry antibody staining. But I am losing more than 40% cells after centrifugation. Does anyone has ever calculated the number of cells with & without centrifugation? I have a window for 5-10% cell loss. Please guide me
Dear Sachin Kumar Singh Chuahan ,
I think 500 g is optimal. The time depends on your volume; for a 15 ml Falcon tube, 10 minutes will be enough. Because the pellet is not very strong, cell loss depends on the care of removing the supernatant. You can increase to 1000 g, if removing the supernatant is difficult.
Best wishes,
Rustem Uzbekov
For my samples I use a 10mL tube for 5min at 400 or 500xG. While I have not directly assessed the supernatant , I believe the white cells are close to 100% isolated in the pellet. I recommend too a conical shaped base to the tube
Rustem E Uzbekov Thank You for replying to my question. I have tried several centrifugation speeds to collect cells. Currently, I am working on a functional assay where I transfer cells from well plates and centrifuge it. The volume in the tube usually varies from 1 ml to 1.5 ml. After centrifugation, I tried tube flipping method to discard supernatant and suction method as well. But I am losing 50-70% cells almost all the time. Even I also added 10% BSA + PBS solution as a washing buffer to enhance the collection of cells but It's not happening. Do You have ever count cells after centrifugation comparing to Control?
Kevin Marron Thanks Kevin. I have tried conical Eppendorf as well as compare to FACS tube but still result are same. During the centrifugation, the other cells which are adherent cells that come down easily and hardly loose 5-10% but I am finding very much difficulty in collecting these pNK cells. Meanwhile, I have already tried 100-1000g over 1-5 minutes. But still, results are the same.
If you lose cells because they attach to the walls of the tube, then maybe you should try glass siliconed tubes? I used the same for blood cells.
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