Hi Nilofar.

I think using trypan blue as a dye in a microplate cell viability assay is the wrong reagent. The cells have to be analyzed in a maximum of 30 minutes, otherwise you will observe an increase of dead cells due to toxicity of TB.

Usually such assays are performed in a microplate reader using f.e. BrdU, or other reagents, depending on your experimental setup.

For manual counting you can probably use acridin orange/propidium iodide. AO stains all cells, PI stains only dead cells. You could theoretically stain your cells in the plates and then count them using a fluorescent microscope. This is the time-consuming method.

For more information feel free to visit the following links:

- https://www.bmglabtech.com/cell-viability-assays-measure-how-happy-your-cells-are/

- https://www.sigmaaldrich.com/technical-documents/articles/biofiles/cell-viability-and-proliferation.html

- https://www.rndsystems.com/products/cell-viability-growth-assays-and-reagents

- https://www.thermofisher.com/de/de/home/life-science/cell-analysis/fluorescence-microplate-assays/microplate-assays-cell-viability.html

Good luck

Marcia Gasis thank you.

I would treat your cells with the drug/inhibitor in the 96 well plates and measure release of LDH by treated versus untreated cells to assess the effect of your treatment on cell viability. You could also stain you cells with the mitochondrial dye, JC-1, and then measure the % of cells that are not stained (dead cells)

Caroline A Owen thank you

E.g. via simple staining and counting via using microscopy and a Neubauer cell counting chamber (see https://bioswisstec.com/products/cell-counting/cell-chip-neubauer-improved-cell-counter-50-pcs/). Or digitally via using a cell counter (e.g. see https://www.intas.de/cell-analysis?gclid=EAIaIQobChMIqdDjgeGW5wIVB893Ch3rgwuHEAAYASAAEgIiCfD_BwE).