3.3. MTT-Cell Proliferation Assay and Morphological Evaluation

Evaluating the cytotoxicity of tested compounds on two cancer cell lines (A549/lung and MDA-MB-231/breast adenocarcinoma) and non-cancerous (MDCK/kidney cells) was performed while adopting the formerly described method with small alterations [70]. Cancer cell lines were propagated within Dulbecco’s Modified Eagle Medium-High Glucose (Cat.#: P0103; DMEM_High Glucose with Na.Pyruvate and stable Glutamine, Biowest^TM, Nuaillé, France), while MDA-MB-231 cells were propagated within RPMI-1640 L-Glutamine medium (Cat.#:12-604F; Lonza-Verviers SPRL^TM, Verviers, Belgium). The culturing media were supplemented via 1% antibiotic-antimycotic 100X (Cat.#: L0010; Biowest^TM, Nuaillé, France) and 10% fetal bovine serum (FBS) (Cat.#: EU-000-H; Seralab^TM, West Sussex, UK). Cells were seeded as triplicates within 96-well plate, at 1 × 10⁴ cells per well density, after being counted and viability checked using the trypan blue staining solution (Cat.# ab233465; Abcam^TM, Cambridge, MA, USA). Seeded cells were permitted to adhere for 24 h under 5% CO₂ and at 37 °C incubating conditions. The assigned compounds were dissolved in 500 µL DMSO affording the stock solution (100 mM) being ready for more diluting within the whole medium to obtain the compound’s final concentrations; 0.1, 1, 10, 100 µM for cell treatments. Notably, the final DMSO-culture medium concentration was not allowed to exceed 0.2% (v/v) [71]. Following 24 h compound-cell treatment, the medium was substituted by fresh one and cells were permitted to develop for 48 h. At four hours prior the end of incubation, 10 μL MTT Sigma-Aldrich^TM (5 mg/mL in PBS 1X without magnesium and calcium; Cat.# 17-516F, Lonza-Verviers^TM, Basel, Switzerland) were added to all wells. Following the complete 48 h incubation, 100 μL DMSO was all added to all wells where they were subsequently centrifuged at 4000 rpm for 5 min allowing the formazan crystals of formazan to precipitate. Color was established and intensities were recorded at 490 nm using Synergy-Neo2^® Hybrid MultiMode Plate Reader (BioTek^TM, Winooski, VT, USA), while subtracting the multi-well plates background absorbance at 690 nm. Percentage cell viability was estimated using the subsequent formula: % cell-viability = (average absorbance of treated wells/average absorbance of controls) × 100.