2.9. Lymphocyte proliferation assay (LPA)

Lymphocytes were isolated from fresh blood donated by a healthy human volunteer, after obtaining informed consent, by Ficoll Histopaque (Sigma) method [30]. Lymphocytes were washed with PBS and resuspended in complete media DMEM along with addition of 50 μM β-mercaptoethanol. Cells were seeded at the density of 5 × 10³ cells in each well in 96 well plates and grown for 16 h at 37 °C and 5% CO₂. Cells were treated with the plant extracts of three different concentrations (100 μg/mL, 10 μg/mL and 1 μg/mL) along with the standards viz., 25 μM celecoxib, 10 μM indomethacin and 10 μM NDGA in duplicates and incubated for 24 h. Cells treated with Con-A (4 μg/mL) served as positive control and without Con-A served as control. After 24 h, 20 μl of MTT (5 mg/ml in PBS) was added in each well and incubated for an additional 3 h at 37 °C. After incubation 50 μl of DMSO was added in each well to dissolve the formazan crystals. Absorbance was read at 570 nm in BioTek Synergy Mx multimode reader. The percent growth of lymphocytes was calculated using control as reference.