Centrifugation speeds for cells. - (Feb/05/2013 )
What speed do you guys use to spin down your cells? Is there a rule of thumb for the different types of cells in terms of centrifugation speed?
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.
Thanks
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 ?
Tabaluga on Tue Feb 5 19:12:56 2013 said:
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.
For spinning live cells do not exceed 300 RCF (relative centrifugal forces also known as "g"), it is best to do it as low as possible, I routinely use 100 RCF for 5-10 min and it works fine for all the cancer cell lines that I work with.
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.
Just looked it up, my 2400 rpm correspond to 600 g, apparently....
I will try 100, 200, and 300 RCF for 5 mins and see if there is any significant loss of cells. My 3000 rpm equals to 800 RCF.
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.
Dear Guys,
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:
http://www.endmemo.com/bio/grpm.php
http://www.geneinfinity.org/sp/sp_rotor.html
then I got the required number in G or in RPM
great links, thanks. Some centrifuges have also got the advantage that you can switch between both values on the display, thereby detemining very quickly the relation between their rpm and g.
Just to throw in my 2 cents. In a neuro lab we spin at 90g for glia, and 160g for neurons in 15ml falcon tubes for 3 minutes.
Considering neurons are pretty small I doubt you'd need to go much faster.