**3. Determination of cell viability by trypan blue exclusion method**

Trypan blue is a dye used to determine the viability of a cell. Living cells exclude the dye, and dead cells will take up the blue dye. The blue stain is easily visible, and cells can be counted using a light microscope. The reactivity is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells that exclude the dye are viable. When the cells are confluent, remove the cell media through aspiration, and add 5 ml of PBS swirl and aspirate. Then add 2 ml of trypsin/EDTA, and swirl to cover the monolayer of cells. Incubate for few minutes at 37°C. To remove the cells, strike the side of the plate or the flask with the palm. Check under a microscope to ensure that all the cells are dislodged. Add 8 ml of cell media containing fetal calf serum (FCS) to the cells containing the culture flask. The FCS will neutralize the action of trypsin. Transfer the cell suspension to a sterile centrifuge tube, and centrifuge the cell suspension at 1000*g* to pellet the cells. Wash the cell pellet twice with PBS. Resuspend the cell pellet in appropriate volume of PBS or cell media. Dilute 10 μl of cell suspension, and place 10 μl on a hemocytometer, and count the cells under a microscope. There are grid markings on the hemocytometer that can be seen under magnification. Count the cells in all four other quadrants of the grid. Divide this number by four to determine the average number of cells in one quadrant. To calculate the number of cells, multiply the average number of cells per quadrant by dilution factor. Multiply this number by 10,000 to calculate the number of cells in 1 ml of suspension. The equation is as follows: average number of cells per quadrant C dilution factor C 10,000 = number of cells/ml. To calculate the total number of cells, multiply the number of cells per ml by the volume (ml) of the cell suspension.

Calculating the % of viable cells: The cells (10,000) are suspended in 500 μl media and treated with varying concentrations of drug and incubate for 24 h. Centrifuge at 1500 rpm for 10 min. Discard 400 μl medium. Resuspended the pellet in the remaining medium. Mix 0.5 mg of trypan blue in 1 ml PBS. Take 10 μl of cell suspension and mix with trypan blue solution. Incubate for 5 min at room temperature. Count the numbers of unstained cells on the hemocytometer under a microscope. As mentioned above the dead cells will take up the trypan blue stain. First count the blue cells in the field and then white cells. Count the total number of cells. The percentage of viable cells can be determined by dividing the number of unstained cells by the total number of cells and multiplying by 100. The equation is as follows: % of Cytotoxicity = [No. of blue cells/Total no. of cells] × 10.
