Is 1500rpm for 5-10 minutes not sufficient to pellet down HeLa cells? Why couldn't I see any pellet? Please answer
I would not recommend centrifuging your cells during the resuscitation process. Freezing and the high concentration of DMSO typically used for cryopreservation are stressful enough.
Instead, transfer the thawed cells directly to 10 ml of media in 10 cm plate. Allow the cells to recover overnight, and then change their media to remove the DMSO.
Thank you I will keep that in mind. But could you tell why the pellet was not visible after centrifugation at 1500rpm for 10 minutes? Should I have increased the rpm? John Hardy Lockhart
You should be able to see a pellet if you had a sufficient number of cells (generally 1x106 is a convenient number to freeze down). Otherwise, I would suspect that the cells were somehow damaged/lysed during the freezing process.
I recommend pelleting cells at 300xg for 5 minutes. That's a bit less than 1500 RPM on swing bucket rotors that I've used, but I can't say for sure what that would convert to on your centrifuge.
Yes, I suspect that there may not be sufficient cells and from here onwards shall try something like 1200rpm for 5-7minutes. Thank you again for your advice.
Hi. The best way to revive the cells is always thaw them and immediately add the 1ml medium in a 60mm dish. DMSO will not harm your cells when you added to the culture medium. Once you are sure that dish is 80% confluent, you can trypsinize and centrifuge before placing the cells back in the dish. Also -80 is not a good place to store the cells for a longer period of time. it is always better to place them in liquid nitrogen.
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