Vitabright 48 vb48

NRK-52E cells (1 × 10⁵ cells/well) were pretreated with 80 μg/mL SCAP-ex or 10 μM losartan in 24-well plates for 30 min. Next, 15 μM cisplatin was added and the cells were cultured for another 24 h. The cultured cells were then centrifuged at 1500 rpm for 5 min. The supernatant was removed and the precipitate was suspended in 200 μL of PBS. The suspension was mixed continuously while 800 μL of cold ethanol was added, and the cells were stored at −20 °C overnight. The thiol levels were measured using VitaBright-48 (VB48, ChemoMetec A/S, Lillerod, Denmark) and the number of dead cells was determined using propidium iodide (PI, ChemoMetec A/S, Denmark), according to the instructions provided by the manufacturer of the cell-vitality kit (ChemoMetec A/S, Denmark). The results were obtained using a NucleoCounter NC-250 and the NucleoView software (ChemoMetec A/S, Denmark).

The cell viability was determined using Nucleo Counter NC-3000 (ChemoMetec, Copenhagen, Denmark). Prior the experiment MCF-7 and MCF-7^shPRODH/POX cells were cultured in six-well plates at 1 × 10⁵ cells/well with 2 ml of growth medium. After 24 h incubation of the cells in glutamine-free DMEM with or without GlyPro, medium was discarded and the cells were rinsed three times with phosphate buffered saline (PBS). Then the cells were harvested, washed and stained with VitaBright-48 (VB-48) (ChemoMetec), acridine orange (AO) (ChemoMetec), propidium iodide (PI) (ChemoMetec) and analyzed using NC-3000 cell counter.

Avelox solution for infusion (1 bottle of 250 ml containing 400 mg moxifloxacin as hydrochloride) was obtained from Bayer Healthcare Pharmaceuticals Inc. (Germany). Growth medium DMEM, penicillin G, amphotericin B, neomycin sulfate, fetal bovine serum (FBS), and trypsin/EDTA were purchased from Cytogen (Poland). Cell Proliferation Reagent WST-1 was produced by Roche GmbH (Germany). Staining reagents: DAPI (1 µg/ml), 0.1% Triton X-100 in PBS; VitaBright-48 (VB-48), propidium iodide (PI) and acridine orange (AO) solution; JC-1 (200 µg/ml in DMSO); Hoechst 33,342 (500 µg/ml), PI (500 µg/ml); NC-Slide A2 and A8; Via-1-Cassette were obtained from ChemoMetec (Denmark). Annexin V-CF488A conjugate and Annexin V binding buffer were obtained from Biotium Inc. (USA). Antibodies: anti-Mcl-1 (4572, polyclonal antibody that detects an endogenous level of human Mcl-1, source: rabbit), anti-MITF (D5G7V, monoclonal antibody that recognizes an endogenous level of total MITF protein, source: rabbit), and anti-GAPDH (14C10, rabbit monoclonal antibody) were obtained from Cell Signaling (USA). Anti-Rabbit IgG (A154), RIPA Buffer, Tween-20, and PVDF membranes were purchased from Sigma-Aldrich Inc. (USA). Pierce BCA Protein Assay Kit, penicillin G, amphotericin B, and ECL Western Blotting Substrate were purchased from Thermo Fisher Scientific (USA).