I use density gradient centrifugation with Percoll to extract bacteria from the bentonite sample. It is working quite well, but I need to remove the Percoll from the bacterial extract at the end of protocol, because it somehow interferes with the Life/Dead stain I need to use. Do you have any hint for that? In the Percoll protocol I found it can be washed from the cell culture by the repeated slow speed centrifugation at 200xg in PBS solution, but this did not work at all. Actually, I do not understand the physics behind this particular step, such slow speed can hardly have any effect, even bacteria in the water solution will not sediment at such slow speed. Do you have any suggestions? Thanks.
Our protocol for removing percoll from microglia we isolated originally called for a 220 rpm for 7mins, but we found that no pellet formed, similarly to what you describe (we took 2 mL of the percoll/cell solution and diluted it in 10mL of HBSS). We ramped the speed up to 1800rpm and did get a pellet and successfully grew the cells. Try a higher speed and see if the cells remain viable! I hope this helps!
Thanks Elliot. If I understood it well, the Percoll formed kind of pellet at higher speed (1800rpm), so you just take the supernatant, which included cells only. Is that correct? You do it in a single step? Or you repeat the process several times? Thanks a lot, I will try.
I see... We remove the cells of interest in the percoll gradient and dilute that down before the centrifugation. It is a one step centrifugation and has worked for us!
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