In MTT cell proliferation assay, after seeding cells (eg.HepG2) in 96 well plates (24h) and treating cells with drugs under study and incubation for 48hrs, sudden cell death occurs, including untreated controls. Why? How do I solve this problem?
Hi , i think the cells was 100% confluence and after that cells detached , try to minimize cell concentration
Hi Dear, HepG2 is very fast growing cell line, especially when it is healthy, thus u need to reduce cell concentration (cell number per each well) and serum concentration as well to about (5%). On the other hand, seed at early morning (8.00 am), then treat after 6 hours (2.00 pm). No need to wait overnight, as it is very friendy and easy attachable cell line. Wish u all the best and TQ
Seed at 1000 to 2000 cells per well if u have to jeep them for 48 hours. Maintain with10% fbs at 5% co2....
So how did you judge cell death, if no detachment occurred? Cells did not accumulate MTT? The guess by Rasha that they had reached confluence (and then stopped proliferating) stills sounds the most plausible to me. They may just have arrested metabolism at this point (which is what you indirectly measure with MTT production). At any rate, I can only suport the suggestions to seed at lower initial density, try and you will see.
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