2.7. Evaluation of In Vitro Anticancer Activity Assay

The anti-proliferative effects of TCE, AgNPs and TCE + AgNPs were determined, using the MTT assay [23] on breast cancer (MCF-7) cell line procured from National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), containing 10% Fetal Bovine Serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin, in 5% CO₂, at 37 °C, until confluent. The cells were trypsinized with 0.05% trypsin-EDTA solution for the haemocytometric cellular viability screening before 10,000 cells/well were plated and incubated in 5% CO₂ at 37 °C, until confluent. The treatment was carried out at the TCE, AgNPs and TCE + AgNPs concentrations of 2.5, 5, 10, 20 and 400 µg/mL. The cells were fixed using 5 mg/mL of MTT reagent per well, after 24 h of treatment, and incubated at 37 °C for 1 h, followed by centrifugation for 5 min at 3000 rpm. The plates were then wash with distilled water to remove the excess dye and air dried. 100 μL of DMSO was added on to the plate, to solubilize the formazan crystals formed, and OD was read at 570 nm. % inhibition was calculated using the below Formula (2):

The % inhibition was represented graphically and statistically using the Prism 8 statistical analysis tool (GraphPad Software, San Diego, CA, USA).