Hello Fatma Mohamed Fouad

Your cells should be 80% confluent after 24 hours of seeding when you must treat them. At 80% confluency, cells are actively dividing but haven't reached a point where they are overcrowded and experiencing contact inhibition, which can affect the results. This confluency level ensures a significant number of cells are available to interact with the drug and respond in the assay. When you use 80% confluency, the assay can more accurately reflect the drug's impact on cell viability and proliferation because cells are still actively growing.

If you perform the assay at low confluency, there won’t be enough cells to produce a strong signal in the assay. Conversely, if you use cells at high confluency, they might reach a plateau in their growth, and the drug's effect might not be as pronounced.

Best,

Hello Fatma Mohamed Fouad , it would be best to optimize cell numbers: Seed different cell numbers into the same format as your IC50 assay, e.g. 96well plate. 1 column 1000, next 2000, next 3000 cells etc..

Let them attach over night and then grow for how long you would treat your cells (e.g. 72h). Run your cytoxicity assay. You will obtain values where cell numbers are linear to your readout, and - if you used too many cells - values will linear anymore to cell number increase. Choose a cell number from the linear section.

hope that helps

Gaurav S. Soman.

Thomas Mühlenberg do you mean that I should change the number of seeding cells according to the time of treatment? For example, if I detect that cells in a 5000 cell density have reached 80% after 24 hr ( for 24 hr treatment), so should I seed, for example, 3000 cells if I leave the treatment for 48 hr?

Gaurav S. Soman.

This is cell line dependent. And even if another group published a cell number, I would double check in my own lab - that's good practice. And yes, optimize the cell number for each duration of the experiment. So if you want to determinde IC50 after 24 and after 48h, optimize the cell numbers for both durations.

In the example attached I would choose

Thomas Mühlenberg Gaurav S. Soman.