Celltiter glo 3d cell viability assay kit

To measure ALB production, 100 μl of culture medium was collected from each group and quantified using an enzyme-linked immunosorbent assay kit (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s protocol. The total ALB concentration was normalized to the total adenosine triphosphate (ATP) content of each sample using a CellTiter-Glo 3D cell viability assay kit (Promega, Madison, WI, USA).

K1 cells were incubated to form spheroids (~ 7 days) in ultra-low binding 96-well plates for viability assay and in ultra-low binding 24-well plate for imaging with PI staining. Spheroids were treated with RSL3 and Fer1 for 48 h. Viability of spheroids were determined using CellTiter-Glo 3D cell viability assay kit (Promega). To visualize cell death in the spheroids. 20 μg of propidium iodide (PI) was added for 30 min and microscopic images were taken using Keyence Fluorescent Microscope BZ-X800.

TGCT-PDC and TGCT-PDC-R cells were seeded at 10,000 cells/well in 96‐well plates and simultaneously transfected with indicated siRNAs at a final concentration of 30 nM using Lipofectamine RNAiMAX reagent (Invitrogen). Six hours after transfection, medium was added to increase the concentration of cisplatin to 0.2 µM. Cell viability was measured by quantitation of intracellular ATP content using CellTiter-Glo 3D Cell Viability Assay Kit (Promega) at 96 h after transfection. After 30 min of cell lysis, chemiluminescent values were measured using the TriStar2 S LB 942 Multimode Reader (Berthold Technologies). NEC8 and NEC8-R cells were seeded at 2,000 cells/well in 96‐well plates and 24 h later transfected with indicated siRNAs at a final concentration of 10 nM using Lipofectamine RNAiMAX reagent. Twenty-four hours after transfection, medium was added to increase the concentration of cisplatin to 1.0 µM. At 72 h after transfection, 10 μL of a Cell Count Reagent SF, a reagent containing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) (Nacalai Tesque) was added to each well and the cells were incubated for 4 h at 37 °C. Absorbance of the plates was read on Multiskan FC Photometer (Thermo scientific) at a wavelength of 450 nm.