**6.11 Preparation of drug dilutions: (serial dilution method)**

Firstly 50 mg/ml stock solution was prepared using 100% DMSO solution. From this prepared stock solution various desired final concentrations like 62.5, 125, 250, and 500 μg/ml of test compound solution was prepared as follows:

The dilution factor was 2 for the MTT assay.

For 500 μg/ml: 10 μL sol. Was taken from stock and to this 990 μl media was added.

For preparation 250 μg/ml: From 500 μg/ml solution the 500 μl was taken and was diluted with the 500 μl with media.

For preparation 125 μg/ml: From 250 μg/ml solution the 500 μl was taken and was diluted with the 500 μl with media.

For the preparation of 62.5 μg/ml: From 125 μg/ml solution the 500 μl was taken and then diluted with the 500 μl with media.

Exponentially the well-growing cell lines were collected from a 25 cm2 Tissue culture flask and a stock cell suspension of 5X104 cell/ml was prepared. A 96-well flat-bottom tissue culture plate was seeded with 5 x103 cells in 0.1 ml of F12 medium supplemented with 10% FBS and then allowed to attach for 24 hours. Test compounds were prepared just before the experiment conduction and serial dilution was done with medium to get the working stock of different 200, 100, 50, and 25 μg/ml solutions. After incubation for 24 hours, the cells were treated with 100 μl of test solutions from respective above stocks, and after treating the cells were incubated for 48 hrs. The cells present in the control group received only the medium containing the 0.5–0.25% DMSO. Each treatment procedure was performed in triplicates. After the treatment duration, 30 μL5 mg/ml MTT solution was then added to each well, and the whole was incubated for 3 hours at 37°C in maintained sterile conditions. After the completion of incubation time, the MTT containing media was removed carefully from all wells then formazan crystals were dissolved by adding 100 μL of DMSO. The plate was shaken for 5 minutes on a gyratory shaker machine and the optical density was noted at 540 nm in an ELISA plate reader. The percentage of cell viability was calculated. O.D of each well was read and expressed as % cell death: (Absorbance of control wells- absorbance of test wells/absorbance of control wells) x 100. Results were expressed as the mean ± S.E.M. The O.D values (proportional to cell death) were plotted against the tested drug concentrations and then interpreted [3, 4, 13, 34, 45].
