Hi Dao Chen

You will need to perform a cell density test first. The purpose of this test is to determine the optimal cell concentration to use. Let's say you are going to perform MTT Assay.

The first thing to do is to seed cells at increasing concentrations (e.g., 2,000; 4,000; 6,000 cells per well, etc.) in your 96-wells plate. Carry out the procedure exactly as you would in your actual experiment, except that you do not need to treat the cells with your compound.

Let’s say that in your experiment, you treat the cells 24 hours after seeding and then incubate them for 24 hours with your treatment. For the density test, you would similarly seed the cells, and then after a total of 48 hours (24 hours post-seeding + 24 hours incubation time), perform the MTT assay to measure the OD value at each cell density.

You can then select the seeding density that yields an OD value within the optimal linear range for MTT. ATCC, recommended formazan absorbance for MTT assays between 0.75 and 1.25. You can use that as a reference or lookup for papers that use the same cell lines as you are about to use.

The same principle applies if your treatment condition involves a longer incubation. For example, 48 hours. In that case, perform the MTT assay 72 hours after seeding (24 hours post-seeding + 48 hours incubation).

Once you've determined the optimal seeding density, you can use that concentration in your actual MTT assays. The goal is to identify the cell number that will give an OD reading in the linear range by the end of your treatment. This also helps to avoid overcrowding, which can lead to cell death and affect your results.

If you're using kit, I think they should've outline the protocols for this.