before cell sorting, the pellet is very obvious, centrifugation rate is 800g. However, I can see nothing after cell sorting with the same centrifugation rate. Have I lost most of my cells? Should I use larger centrifugation rate? does anyone have the same problem?
Hi Mei,
If you want to see the cell pellet with your eyes, more than 10^6 cells, at least I think, are needed. After sorting, the cell number must be reduced. the colourless and transparent buffer solution may also enhance the difficulty of visualizing the cell pellet.
Best,
Xiangchun
Hi Mei,
How many cells did you start with? How many were sorted to the fraction you're work with (i.e. the one your spinning down)? A cell pellet gets more or less "invisible" if you have small cell numbers. Are you working with mammalian cells? 800g sounds quite high to me. I usually use 200-300g. Also be careful when discarding supernatant you may lose precious cells e.g. by decanting FACS tubes.
Hope that helps
Hi Mei,
Are you sure your cells survive the sorting procedure? Dead cells tend to notoriously stick to the sides of tubes when spun down, rather than forming a nice pellet. Maybe check a small sample with trypan blue before spinning?
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?
Ahh, unfortunately i did not get any RNA from my cells, so i suspect that there is a problem at least in my experiment - Thank you for your reply.
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