I am doing a viability test to see the application of Calcein AM and EthD-1 on different platforms. Say, I have sub-populations of live and dead MDA-MB-231's in 2.5 mL of ECB (total cell density: 6 million/mL). Now, I add 2 uL of Calcein AM and 4 uL of EthD-1 to this mixture and incubate it for 15 mins.
If I use this sample directly out of incubator (without pelleting and washing), this seems to give a very high background/noise/false signal under different conditions. But, I am also worried if I would lose the dead cells if I pellet and wash it post-staining.
Does centrifuging remove all the dead cells? If not, what should be the optimum centrifugation parameters? Can I get rid of this high noise caused by stains through some way?
Thanks!
Hello, Manibarathi!
According to my experience, centrifugation does not remove dead cells. The optimal parameters for cancer cell lines are about 100-200 g for 5-10 miutes. To be more confident about cell harvesting you can use some tips:
1) The centrifugation speed can be increased even more (up to 800 g), but you should probably test these conditions before the main experiment to ensure that the chosen parameters does not affect viability.
2) Use bucket rotor vith V-bottom tubes or plates.
And there is one more thing to remember about viability assay of adherent cells. The usual way to resuspend it is to remove culture media and replace it with dissociation agent like trypsin or EDTA. But if you discard the culture media you can loose dead cells, that often detach from plastc surface during cell death. I am not sure if it is urgent for MDA-MB-231 cells, and I do not know how you detach it, but this general consideration might help.
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