Are the cells are in a single cell suspension before plating and aggregate later? This clumping leads to the increase in cell death and debris formation. You can try increasing serum concentration. If you are using Trypsin you can try using lower concentrations or use gentler enzymes like Accutase

Ashutosh Acharya The cells were in a single-cell suspension before plating, and they later aggregated as you said. I have done my best to evenly distribute the cells, but aggregation still occurred. Even with cells from the same passage, some plates were plated more evenly, but cell death still occurred, albeit delayed compared to the aggregated cells. I will try increasing the serum concentration. Thank you very much for your assistance!

It's unfortunate to hear that you're experiencing difficulties with culturing 22RV1 prostate cancer cells. Sudden cell death and changes in cell growth behavior can indeed be challenging to troubleshoot, but there are several potential factors to consider:

If the problem persists despite troubleshooting efforts, consider obtaining a new vial of 22RV1 cells from a reputable cell repository to rule out issues with the cell line itself. Additionally, consider performing additional assays or tests to further characterize the cells and identify underlying causes of the observed phenotypes.

Arvind Kumar Shukla First of all, thank you very much for your suggestions. However, to be frank, I find your advice too general. It's difficult to address my specific issue based on your response, as all the problems encountered with the cells could be explained using your suggestions.