2.7.2. Cell Viability

Cell viability was measured using the method as described by Berrington and Lall [24] using the 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) Cell Proliferation Kit II (Merck Millipore, USA). The HepG2 cells were seeded at a concentration of 2000 cells/mL, whereas the remaining cell lines were seeded at a concentration of 1.0 <sup>×</sup> 106 cells/mL in 96-well plates (100 μL) and allowed to adhere for 24 h. The *S. perfoliata* extract was prepared at stock concentration of 20 mg/mL, serially diluted and added to the 96-well plate at final concentrations ranging from 1.56 to 200 μg/mL. Controls included a 2% DMSO vehicle control, cells grown in medium only and Actinomycin D at final concentration ranging from 3.9 <sup>×</sup> <sup>10</sup>−<sup>4</sup> to 0.05 <sup>μ</sup>g/mL. Cells were incubated for a further 72 h with the respective samples and controls. Thereafter, 0.05 mL XTT (0.3 mg/mL) was added to the cells and incubated for 2 h where after the absorbance was measured at 490 nm (reference wavelength of 690 nm) using a BIO-TEK powerwave XS plate reader. Blank plates were included, which were prepared in the same manner as mentioned above, without the additional of cells, to allow for color compensation of the samples. The percentage viability was calculated as follows and the IC50 value was then calculated.

$$\text{Percentage viability} \left( \% \right) = \left[ \left( \frac{\text{Abs}\_{\text{sample}/\text{positive}}}{\text{Abs}\_{\text{control}}} \right) \times 100 \right].$$
