Accutase solution

Human iPS cell lines (201B7 and 1231A3) were obtained from the Center for iPS Cell Research and Application (Kyoto University). Human iPSCs were maintained on LM511-E8 (iMatrix-511; Nippi; 0.25 μg/cm²) in an uncoated manner, along with StemFit medium (Ajinomoto), in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. At passage, the cells were dispersed in Accutase solution (BD Biosciences) for 5 min at 37 °C. Human iPSCs were seeded in a culture medium containing 10 μM Y-27632.

For assay of cell viability, B16-F10, H1703, A549 and NIH-3T3 cells were plated in 96-well round-bottomed plates at a density of 2 × 10³/well and incubated for 72 h with various concentrations of nintedanib. Cell viability was then assessed with the use of a CellTiter-Glo 3D Luminescent Cell Viability Assay (Promega). For assay of cell proliferation, B16-F10 or NIH-3T3 cells were seeded in 10-cm dishes and exposed to 0.5 or 1 μM nintedanib for 24, 48 or 72 h. The cells were then washed three times with ice-cold PBS, collected with the use of Accutase solution (BD Biosciences), and stained with the use of a Zombie Fixable Viability Kit (BioLegend) for discrimination of live from dead cells. The living cells were counted with a flow cytometer (LSRFortessa X-20, BD Biosciences) and normalised by those in untreated cultures.

A-375 cells were seeded in 12-well plates using 100,000 cells per well and left to adhere for 24 h. Cells were treated with 50 nM salt 1-3C or salt 1-8C for 30, 60, 90, and 120 min. Cells were harvested with accutase solution (Sigma-Aldrich), washed twice with phosphate buffered saline (PBS), and resuspended in PBS. Uptake of both compounds was measured by the fluorescent properties of the compounds detected in the channels for APC (allophycocyanin) (Ex-Max 650 nm/Em-Max 660 nm). The fluorescent signals were evaluated by BD FACSVerse™ (BD Biosciences, Franklin Lakes, NJ, USA) using Cell Quest software (BD Biosciences). All samples were analyzed in triplicate.
Mitochondrial membrane potential was analyzed using JC-1 dye (Thermo Fisher Scientific, Waltham, MA, USA). A-375 cells at a concentration of 250,000 cells per well were cultured overnight in 6-well plates. Cells were treated with 2000 nM salt 1-3C and 5000 nM salt 1-8C for 15 min, 2 h, 4 h, and 24 h or were untreated (time 0). Cells were harvested with accutase solution, washed twice with PBS, resuspended in PBS with 5 µg/ml JC-1, and measured by FACSAria^TM (BD Biosciences). Data were analyzed in triplicate by FCS Express version 4.0 (De Novo Software, Glendale, CA, USA).