I want to perform MTT assay and for that cell density plays a very crucial role. How to determine cell number to be seeded for a particular cell line.
The sensitivity of MTT assay depends on several factors such as metabolic activity of the cells (cell type), cell number, proliferation capacity, duration of the treatments (if you want to check the viability of cells in presence of some drugs) etc. I would suggest to culture different cells densities for a specific duration of the planned experiment and do MTT assay to find out the proper density where there is a minimal cell death and and unsaturated MTT absorption. If you are going to use a drug that causes cell proliferation or increased metabolic activity, I would suggest to have lower cell densities for your experiments. I hope this helps. Good luck
In performing an MTT assay is crucial that your cells have in the well enough space to be actively proliferating for all the duration of the treatment, in order to observe a decrease in the proliferation only when a proper amount of cytotoxic drug is present. If cells density reaches a plateau during treatment, the subsequent absorbance/fluorescence reading will generate a curve that will not exactly reflect the cytotoxic effects of the drug you employed, and IC50 calculations will be distorted. You should first have a look at your cells doubling time in basal conditions and perform a little growth curve assay. Thereafter you should carry-out few MTT assays with different cell densities for the maximum duration of what will be the real assay. Then measure absorbance/fluorescence and plot the data to generate a log-dose response (where actually the dose is the amount of cells). Compare curves of the first 24hrs growth with the last point of growth. You will then operate below the cell densities that reach a plateau at the last point of growth.
As Dr. Budida suggest, keep in mind that cell density should be lower if you employ drugs that enhance proliferation, as plateau will be reached sooner.
By the way, just as a suggestion, I usually seed between 5.000 and 10.000 cells per well (for 24-48 hrs usually), but as said, check your cells proliferation rate for peace of mind.
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