2.13. MTT (3‐{4, 5‐dimethylthiazol‐2‐yl}‐2,5 diphenyl tetrazolium bromide) anticancer assay

The phytoextracts were dissolved in DMSO and then filtered with the help of regenerative cellulose filter to avoid contamination. AgNPs were dissolved in water using sonicator and sterilized with the help of ultraviolet radiations. Seven different concentrations (18.5, 37, 65, 125, 250, 500, and 1000 μg/mL) of each test sample were made in high glucose DMEM as per by specifications of the experiment.

HepG2 cell line was used in this study. Cells were revived in high glucose DMEM by first thawing the cryovial, then adding it into 2 mL of FBS (fetal bovine serum) and 8–10 mL of high glucose DMEM. After centrifugation at 1000 g for 8 min, the pellet was plated in 75‐mL cell culture flask and incubated for 24 h.

2 × 10⁴ cells were plated in 24 wells (seven doses, one control, each in triplicate) of the 96‐well plates (each for one test sample). After 24 h of incubation, different doses of test samples were added to the cells in each plate as in the order of: Control, 18.5, 37, 65, 125, 250, 500, and 1000 μg/mL.

Next day, MTT assay was performed in which 100 μL of MTT solution (5 mg/mL in 1X PBS) was transferred to each well after washing with PBS. The microplates were wrapped under aluminum foil and kept in the incubator at 37°C for 3 h. After incubation, the media were aspirated carefully so that no Formazan crystals were removed from each well. Then, 100 μL of DMSO was added to each well. The 96‐well plates were shaken for 10 s so that the Formazan crystals were dissolved resulting in the purple‐colored solution. Spectrostar Nano (BMG LABTECH) microplate reader was used to measure the optical density at 570 nm and 630 nm (Zughaibi et al., ²⁰²²).