I need to do cytotoxicity study of some anti-cancer drugs on normal cells HEK293T. But the cells are detaching from the well after treatmet with MTT. How to solve this problem?
Hello Navas Shereef Ellyan Majeed,
HEK293T cells have a natural tendency to detach from the substratum, especially when they are overconfluent or with little agitation because they have a relatively weak ability to adhere to the culture plate surface. So, whenever you perform MTT assay with HEK293T cells you have to be really careful.
Incorporating the following measures may help solve your problem.
1. Do not allow cells to become too crowded (overconfluent), otherwise they are likely to detach in sheets or clumps.
2. Handle these cells gently because even slight agitation or pipetting can cause HEK293T cells to detach. So, be extra careful while washing, changing media, or pipetting HEK293T cells. Avoid vigorous movements.
3. HEK293T cells are temperature-dependent for attachment, so maintaining a consistent temperature of 37°C is crucial. So, always use pre-warmed medium and reagents to maintain optimal temperature and prevent temperature shock.
4. HEK293 cells need calcium and magnesium to attach to regular TC-treated cell culture plastic. Washing them with PBS, which doesn't contain calcium or magnesium, can lead to detachment. So, use PBS or other physiological buffer with added calcium and magnesium.
5. Pre-coat your culture plates with poly-l-lysine, collagen, or other adhesion molecules to improve cell attachment.
The above tips will help!
Best,
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