Would it be OK to culture cells in 96 well plates for over 72 hours? We need to run an experiment which involves cell seeding then treatment after 12 hours. The cells should then be incubated with the drug for 72 hours.
We use HEK and HEPG2 cells, starting cell density 25,000.
Thanks
Hey Salma,
we cultivate our cells (MEFs, bone marrow macrophages, peritoneal macrophages) also 72 hours, if the experimental setup demands it in 96-well plates. Of course you have to be careful, if you culture a dividing cell line. Macrophages, which do not undergoe division, can be cultivated for 10 days with DMEM + 10% heat-inactivated FBS. I think the well size does not matter in this context. Cells start dying, when they are not happy with the medium or if they reach the maximal point for suriving outside the body.
All the best,
Marc
Duplication time for HEK 293 is about 34 hrs , so you should consider consumption of media (which is containing drug , can't be changed ) and starting seeding density which be suitable to the max number could be seeded in a 96-well to avoid overgrowth of your control cells or cells under treatment with ineffective dose ( in this case you wouldn't be able to measure the viability after treatment accurately .
The capacity of 96 well plate is 1-1.5 lakh cells grow easily. Accordingly you can set a reaction time and number of cell seeded. As my experience, it will be better to seed 5000-10000 cells/well for 72 hrs for HEk and HepG2.
Thank you
You should have a cell number that can grow in the exponential phase until the end of your assay. For 3 days MTT assay, your cell’s growth will start slowing down due to medium and well limitations. Thus, MTT reading will have a false error. Hence, you are advised to select your seeding density based on cell number titration.
For Hep G2 MTT assay (96 well plate), I usually seed 10000 cells/well for 24/48 hr and 6000 cells/well for 72 hr assay.
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