We used the Promega MTT assay a few years ago. From my understanding this assay is usually plotted with absorbance on the Y-axis. Meaning the results are presented as relative changes from control, over time or between treatments.

In these instances we would often perform a negative control (untreated) and a positive control (lysed or other method of cell death). These are not a standard curve, but are a common approach to determine level of cell death from 'healthy' to 100% cytotoxicity.

If you want to find out whether there's a linear increase in signal (i.e. cell viability) with increasing cell numbers, here's what you need to do.

From your question, I believe you'd like to find the optimum cell count and incubation period for your cell type. If that's the case, here is what you need to do:

1. Harvest your cells and measure the concentration.

2. Prepare a serial dilution of your cells, typically from 1 x 103 to 1 x 106 cells/ml, and seed them in duplicates or triplicates. Also make sure you prepare controls.

3. Incubate the cells. You could incubate from 6-24 hours depending on your cells. I find that most cells do okay with a 12 hour incubation time.

4. Then perform MTT by adding the MTT reagent which depends on the kit you have purchased. After obtaining the optical density, you can go ahead and plot your standard curve.

I only do this once for every cell line to find the optimum cell count for seeding.

I hope I have answered your question. Cheers.

Hi @ Lewis Williams

Thank you for your response, I am this time years old when I found that I need to press the question title so I get to see the answers.

these are helpful tips with the control, much appreciated.

Many thanks

Asma

Hi @ Hossein Jahedi

Thank you for your response, much appreciated.

According to this, what do you suggest for the positive control. I can use Mitomycine treated cells to stop them from proliferating and use this as negative control.

Many thanks

Asma