**4. Viability assay in normal cells**

Attempts are pursued to develop drugs that are nontoxic to normal cells; meanwhile toxic to cancer cells can be considered as good anticancer agent.

### **4.1 Isolation of lymphocytes from whole blood**

In 1968, Boyum [21] described methods for the isolation of mononuclear cells from circulating blood and bone marrow. The solution contains Ficoll and sodium diatrizoate, adjusted to a density of 1.077 ± 0.001. This medium facilitates rapid recovery of viable lymphocytes from small volumes of blood on centrifugation. It is usually employed as the initial isolation step prior to enumeration of T, B, and null lymphocytes. In brief, 3 ml of blood will be taken in heparinized test tube. About 5 ml of PBS will be added and mixed well by inversion. About 3 ml of Ficoll Hypaque solution was added in a conical centrifuge tube. Carefully layered 8 ml of blood-PBS mixture on to the Ficoll Hypaque, keeping the tube in a slanting position. Centrifuged at 2000 rpm for 30 min. The opaque interface containing mononuclear cells was aspirated and transferred into a clean conical centrifuge tube with a Pasteur pipette and discard the upper layer. About 10 ml of PBS solution was added and mixed by inversion. Centrifuged at 1500 rpm for 10 min and the supernatant was discarded. Resuspend the pellet with 5 ml PBS and centrifuge at 1500 rpm for 10 min. Repeat the step thrice and resuspend the lymphocyte pellet in 500 μl PBS.

#### **4.2 Lymphocyte viability assay**

In vitro experiments with compounds can be tested in lymphocyte using lymphocyte viability assay. The lymphocytes were aspirated from the gradient plasma interfaces and washed twice in PBS, and then the final cell pellets will be resuspended in RPMI-1640 medium containing 10% FCS and 100 μl streptomycin, and 100 μl fungicide can be added to avoid contamination; pH 7.4 is ideal. Cells can be harvested, counted, and seeded (5 × 103 cells/well in 100 μl) in 96 well titer plates (Axygen). PBS will be added to the outer wells (200 μl/well). After 24 h of incubation at 37°C in 5% CO2 to allow cell attachment, the media will be

removed. Cells will be treated with varying concentration of compounds diluted with medium. The plates can be incubated for 72 h. After incubation, the media will be removed without disturbing the cells, and to each well, 100 μl of 5 mg/ml stock solution of MTT will be added, and the plates can be incubated for 2 h in the dark at 37°C in a CO2 incubator. About 100 μl of lysis buffer will be added to each well, and the plates can be further incubated for 4 h in the dark in a CO2 incubator, and absorbance can be read using ELISA plate. Three replicates will set up for each concentration.
