Nitroblue tetrazolium chloride

After A549 cells were treated with metformin and/or tenovin‐6 for 48 hours, cells were trypsinized. Cell suspension was mixed with 0.3% soft agar in growth medium and layered (1000 cells/well in 6‐well plates) on top of 0.6% base agar with growth medium. After 2 weeks, cells were stained with a nitro blue tetrazolium chloride (Sigma‐Aldrich) solution (1 mg/mL in PBS) overnight at 37°C. Colonies containing more than 50 individual cells and those with diameter greater than 0.5 μm were counted using Image J software (National Institutes of Health, Bethesda, MD, USA). All experiments were performed in triplicate.

A nitroblue-tetrazolium (NBT; Sigma-Aldrich, St. Louis, MO, USA) assay was used for detection of the intracellular superoxide radical level. This is a colorimetric method conducted by assessing blue formazan deposits, formed by the reduction of the membrane-permeable and yellow-colored nitroblue-tetrazolium chloride (2,2’bis(4-nitro-phenyl)-5,5’-diphenyl-3,3’-dimethoxy-4,4’-diphenylene) diterazolium chloride; Sigma-Aldrich, St. Louis, MO, USA) by the superoxide radicals [40 (link)]. Mouse TM3 cells were pre-cultured at a density of 4 × 103 cells/well in 96-well plates for 24 h. Afterward, cell culture media was replaced by DMEM/F12 supplemented with corresponding doses (from 62.5 to 2000 µg/mL) of Lepidium sativum L. for 24 h and 48 h. Subsequently, the NBT salt was dissolved in DMEM/F12 containing 1.5% DMSO (final concentrations: 1mg/mL) and added to the Leydig cells. After a 3 h incubation (37 °C; 5% CO₂ and 95% atmospheric humidity), the formed blue deposits were solubilized with 2M potassium hydroxide (KOH; p.a. CentralChem, Bratislava, Slovakia) dissolved in DMSO. The optical density was measured at a wavelength of 620 nm against 570 nm as a reference, using a microplate reader Multiscan FC (Thermo Fisher Scientific, Waltham, MA, USA).