Centrifugation speeds for cells.
Posted 05 February 2013 - 10:58 AM
I am having a problem with my cell count and I am not sure if the spinning down process is shearing my cells. Last night I counted my cell suspension with a hemocytometer and it came out to 1.5*10^6 cells but the FACS analysis reported a much lower number (around 10^4 cells). I know I will lose cells during the staining process and during the analysis but going from 10^6 to 10^4 is huge loss. Any advice will be appreciated.
Posted 05 February 2013 - 11:12 AM
Posted 05 February 2013 - 11:22 AM
Hm, I spin my cells down with 1300rpm for 3 min. Later during the FACS staining process I spin them down with 2400 rpm for 3 min, but be careful as this can harm the cells. Interestingly I also observe huge cell loss during the staining process although I never quantified it like you did... Do some of your cell stick to the wall of the reaction tube perhaps ?
Working with epithelia cells I use 3000 rpm for 3mins and working with HSCs the protocol calls for 1500 rpm for 5mins. I don't think my cells are sticking to the wall of the reaction tube but I don't know for sure. I don't know if it is pipetting error, cell count error or the speed of spinning.
Posted 05 February 2013 - 12:30 PM
When working with centrifuges, rpm is a relative measure that has no reference unless you also provide the rotor radius so Wek's 3000 rpm may in fact be lower RCF than Tabaluga's 2400, but we will never know.
Spinning too fast can cause a "smear" of cells up the wall of the tube that you may be missing when resuspending the cells.
Posted 05 February 2013 - 12:36 PM
Posted 06 February 2013 - 10:31 AM
I have actually noticed the line smear on the wall of the tube when using compensation beads but haven't really noticed it when spinning down cells.
Edited by Wek, 06 February 2013 - 10:32 AM.
Posted 17 February 2013 - 03:16 PM
I know this is out of ur discussion, but regarding converting RPM into G or vice verse, I just measure the radius of my centrifugal ring, not the whole machine, just the rotor radius with a ruler or something, put the data on RPM-G converter like these website:
then I got the required number in G or in RPM
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Posted 17 February 2013 - 03:36 PM
Posted 27 February 2013 - 02:59 AM
Considering neurons are pretty small I doubt you'd need to go much faster.