Hey guys,
I have noticed that one day following passaging that instead of distributing evenly across my 10 cm plate that there are large areas that are largely devoid of cells. There are cells in those areas but they form these tight clumps or spheres that are not very adherent. Has anyone seen this before in culture? I passaged from a 75-80% confluent plate that had cells that were well distributed and looked healthy.
Here is how I passage:
Aspirate media -> 1 mL trypsin (incubate 37 C for 3-5 min) -> Neutralize with 8 mL media with FBS -> Pellet 1500 rpm for 5 min -> Resuspend in 10 mL media -> Split at 1:10 or 1:20 -> Rock plate back and forth to mix (no swirling) -> Keep in incubator.
Immediately after passaging, the cells look evenly distributed across the plate. The cells are P27.
Hello,
I have never had those kind of problems with HEK but in anycase you should test your cells for mycoplasms infection.
After centrifugation and aspiration of your tripsin medium, I usually resuspend the cells by pipetting and do likewise in the flask before putting in the incubator. I also never go after P20 (depending on what you're using your cells for of course).
Good luck !
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