2.3. Cell Viability Assay

A clear-bottom 96-well black plate (VWR, #29444-008) was seeded with Caco-2 cells (40,000 cells/well) and grown for 15 days in EMEM media (20% FBS and 1% Pen-Strep) to achieve differentiation of the cells. The MTT assay kit (Abcam, #ab211091) was used to test the cell-viability assay following the manufacturer’s guidelines, as described in the earlier study [11]. For the time-course study, a concentration of 1 mM γ-EV was used for determining the cell toxicity, by incubating it on the Caco-2 cells for 2, 4, and 6 h. For the dose study, the cell toxicity of γ-EV was tested for different doses, such as 2.5, 3, 4, 5, and 10 mM, after incubating the peptide on the cells for 2 h. After the treatment period, the cell media was discarded and replaced with a mixture of 50 μL of the MTT reagent and 50 μL of serum-free EMEM media. After the incubation period of 3 h at 37 °C, the MTT-supplemented media was replaced with 150 μL of MTT solvent. Following that, the plates were incubated on an orbital shaker (200 rpm, 37 °C) (MaxQ 4450, Thermo Fischer Scientific, Waltham, MA, USA) for 15 min, in dark, after which the absorbance of the plate was read at 590 nm (Synergy H1 microplate reader, BioTek, Winooski, VT, USA). The background control readings were subtracted from each of the wells and the cell-viability percentage (%) was calculated using the formula cell viability % = (sample/control) × 100, where the control was cells with no γ-EV treatment, and the sample was the γ-EV treated cells.