After doing a MACS sort and a FACS sort to isolate naive B cells, I've had troubles spinning down my KO cells. I first used the conditions mentioned in the protocol: 400g for 5 min, in 15 or 50ml tubes depending on the volume we had. I couldn't spin the cells down so we ended up spinning them at 1000g for 10 min. I was able to collect the WT cells, but I still didn't manage to spin down the KO cells... Has anybody had that problem before or any idea on what could be the problem ? Thanks
Thank you for your reply. We stained our cells with trypan blue and counted them after FACS sorting. Had they been dead at the time we should have seen loads of dead cells at that point, which we didn't. I used the KO cells I could spin down in my experiment and they looked okay after three days of culture, so I don't think that could be it. We probably should stain some cells with a viability marker next time to be sure though, thank you.
I experienced the same problem, starting with 20 M cells (MNC) and after soring having appr. 600.000 Cells (naive B-cells), 80-90% viable, However, no visible pellet is formed even after 10 min centrifugation at 500 g.
Does anybody has an idea of what could be done? Did you ever find a solution to the problem Hind?
I had that problem back when I was doing an internship at Trinity College, now I'm back in Belgium and I haven't repeated the experiment. So I've never found a solution.
I've had the same problem once, sorting T cells. What happened was that the drop delay wasn't parametered correctly, so the machine was counting drops and correlating it to cells, but those drops were actually empty, which resulted in a much lower cell number than what we expected.
So I wonder if that could've been it. It seems doubtful, because the cell sorter was operated by a specialist and that problem happened every time. But honestly, so far that's the only semi-valid hypothesis I have.
Mette, did you count your cells after sorting ? And if so, did you find the same number of cells between your count and the number the sorter showed ?
If I'm correct, I think you don't really see a cell pellet under 1 million cells ? But the cells might've been there anyway ?
Hi Hind
I sorted my cells using Robosep so it does not show the counting.
I only counted the mononuclear cells on a nucleocounter before freezing (20 M cells/vial), and after the Robosep sorting where i get a negative fraction that contains Naive B cells - When i count them there are 1,2-1,8x105/mL, and viability 85%. I extract RNA directly from the cells, but get extremely low concentration, which is why i still suspect that there is a problem of spinning down the cells.
Anyway, thank you for your reply.
Best
Mette
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