Wst 1 cell viability assay

Human umbilical vein endothelial cells (HUVECs American Type Culture Collection, Manassas, VA, USA) were cultured in a CO₂ incubator using Medium 199 (M199, LabClinics, Barcelona, Spain) supplemented with penicillin-streptomycin (100 units and 0.1 mg/mL, respectively) and 10% fetal bovine serum (complete M199) in T-25 flasks (SPL Life Sciences Co., Ltd.), allowed to grow up to 70–80% convergence and replated by trypsin treatment. Unspecific toxicity of the samples was tested with the WST-1 cell viability assay (Roche Applied Science, Penzberg, Germany), following the manufacturer’s recommendations. Briefly, HUVECs were seeded in 96-well plates at a density of 5000 cells per well in 100 µL of complete M199. After a 24-h incubation at 37 °C, the medium was removed, and 90 µL of fresh M199 were added together with 10 µL of the sample of interest in PBS. HUVECs were placed back in the incubator for 24 h or 48 h. At the moment of reading, 10 µL of WST-1 reagent was added to each well, and, after an incubation of 3–4 h, absorbance at 440 nm was measured with an Epoch^TM microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). For each sample, three different concentrations were tested in triplicates, and each plate contained three seeded wells with 1% bleach (0% viability control) and three wells with 10 µL PBS (100% viability control) as controls.

To determine the cytotoxicity of the treatments used in our experiments, the WST-1 Cell Viability Assay (Roche) was used. Cells were seeded (5.0x10⁴/well) onto a 96-well plate and were allowed to attach overnight. The following morning, the full medium was removed and was replaced with 100μL of serum-free medium containing various concentrations of D-glucose (5mM, 10mM, 15mM, 25mM, 50mM, 100mM). Following 24 hours of incubation with glucose, 10μL of WST-1 reagent was added to each well. The plate incubated for 1.5 hours at 37°C to produce a colour reaction. Absorbance was measured with a Multiskan FC Microplate Photometer (Thermo Scientific, Finland) at 450nm with a reference wavelength of 690nm. Survival was determined by the difference between the absorbances at 450nm and 690nm.

Cell viability was assessed using the Muse™ Cell Analyzer and the Muse™ Count and Viability Kit according to manufacturer’s instructions (EMD Millipore, Billerica, MA, USA; Cat #MCH100102) or the WST-1 cell viability assay (Roche, IN, USA; Cat #5015944001). The plates were mixed on an Orbital Shaker on setting 2 (Bellco, NJ, USA; Cat #7744-20220) at room temperature for 30 min and absorbance was measured at 630 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad, CA, US). MTT cell viability assay (Thermo Fisher MA, USA; cat #V-13154) was performed by incubating culture plates with MTT reagent for four hours at 37 °C, adding SDS lysis buffer, and incubating for four hours to overnight at 37 °C. Absorbance was measured at 570 nm with a plate reader as above.