Dear Dr. Francine Tankeu

I too had experienced the same problem.

HEK293 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.

Incorporate these measures in your protocol, and I hope it may 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 HEK293 cells to detach. So, be extra careful while washing, changing media, or pipetting HEK293 cells. Avoid vigorous movements.

3. HEK293 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.

Regards,

Malcolm Nobre

Dear Malcolm.

Thank you so much for your response and so well-detailed advice and suggestions.

I really appreciate.

dear Francine Tankeu ,

• I had a similar case, and there are multiple reasons this issue can occur.
• You should first check if the incubator is working properly. Sometimes, it may appear to be functioning, but that might not be the case.
• Pay special attention to the following parameters: CO₂ concentration: 5% Temperature: 37°C pH levels
• Additionally, you should consider doing a pre-coating with Lysin

From my experience, this cell lines tend to detach once it grow >80% confluency. So try to passage them before they grew >80% confluence.