Trypan blue staining and viable cell percentage calculation

We prepared a 4% Trypan blue stock reagent by dissolving 4 g of Trypan blue (Merck KGaA, Darmstadt, Hessen, Germany) in 100 ml of double-distilled water and filtering the solution through a filter paper. The stock reagent was diluted to 0.4% with PBS before use. A single-cell suspension was prepared from five groups of cells labeled with 0, 12, 25, 50, and 100 μg Fe/ml MIRB, respectively. An equal amount of 0.4% Trypan blue and single-cell suspension were mixed, and the mixture was incubated for approximately 3 minutes at room temperature. Then, live and dead cells were counted individually under a microscope (dead cells were stained light blue and live cells were colorless). The percentage of viable cells was computed using the following formula: Live cells percent (%)=Total number of live cells/(Total number of live cells＋Total number of dead cells)×100%.