3.2. MTT Cytotoxicity Assay

The colorimetric MTT Cell Proliferation^® Assay Kit (Cat. №. 10009365; Cayman Chemical^TM, Ann Arbor, MI, USA) has been utilized to assess the cytotoxic impact of the investigated compounds on solid cancer cell lines [69]. The cancer cell lines, A-498-ATCC^® HTB-44^™ kidney carcinoma, SCaBER- ATCC^® HTB-3™ bladder squamous cell carcinoma, and HEK-293 normal human embryonic kidney were obtained from American Type Culture Collection. Cells were cultured in 96-well plate, at cell density of 1.2–1.8 × 10⁴ cells/well, using Dulbecco’s Modified Eagle Medium (Invitrogen^TM, Carlsbad, CA, USA) supplemented with 10% (w/v) fetal bovine serum (Hyclone^TM, Marlborough, MA, USA), 10 ug/mL of insulin (Sigma-Aldrich^TM, St. Louis, MO, USA), and 1% (w/v) penicillin-streptomycin (Sigma-Aldrich^TM, St. Louis, MO, USA). Following 24h-incubation period under 5% CO₂ at 37 °C, cells were treated with compounds’ serial concentrations (0.1 µM–100 µM) in DMSO, where the DMSO final concentration in the culture medium never exceeded 0.2% (v/v). Subsequently, the treated microplates were incubated for 48 h at 5% CO₂/37 °C prior to examination under the inverted microscope and proceeding for the MTT assay. The culture medium was discarded and 100 µL of complete medium comprising 5 mg/mL MTT dye were dispensed within every well, then plates were reincubated for 2 h at 5% CO₂/37 °C. Following a complete incubation, dissolution of the resulting formazan crystals was achieved through adding an amount of MTT Solubilization Solution [M-8910] equal to the original culture medium volume. Color intensity within each well was measured spectrophotometrically using the ROBONIK-P2000^® ELISA microplate reader (Bio-Tek^TM, Winooski, VT, USA) at 450 nm while subtracting the background absorbance of multi well plates at 690 nm. The experiment was conducted in triplicates and IC₅₀ values were estimated through linear regression between the logarithm-transformed compound concentrations and corresponding % cell viability = (mean absorbance in test wells/mean absorbance in control wells) ∗ 100, having the response variable slope (four parameters) for all samples.