Ez cytox cell viability assay kit

The effect of cisplatin on the viability of both 2774-control and 2774sh-SOX9 cells was determined using the EZ-Cytox Cell Viability Assay kit (Daeil Lab service, Seoul, Korea) in accordance with the manufacturer’s instructions. Each cell line was cultured in a 96-well microplate at 2.5 × 10³ cells per well in triplicate in a final volume of 100 µL DMEM containing 2.5 µM cisplatin under the conditions described. DMSO was used as the vehicle control. After 0 h, 24 h, 48 h, and 72 h incubation, 10 µL of EZ-Cytox was added, followed by incubation for 60 min at 37 °C and measurement of the absorbance at 450 nm using an ELISA reader, SpectraMax ABS Plus (Molecular Devices, San Jose, CA, USA). For the determination of cell viability in RIPK1siRNA or control siRNA-transfected 2774 cells, cells were seeded at 10,000 cells/well in a 96-well plate, allowed to adhere overnight, and treated with cisplatin for 72 h. Cell proliferation inhibition was determined via Cell Titer Glo (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The detected luminescent signal was used to calculate the percentage of surviving cells for comparison with that of the control.

Proliferation assays were conducted with EZ-Cytox cell viability assay kit (Daeil Lab Service), according to the manufacturer's instruction. In detail, 10³ NIH 3T3 cells were seeded into 96-well plates (100 μl/well, DMEM +10% BCS). After 0, 1, or 3 days, 10 μl of EZ-Cytox reagent was added to each well and incubated for 2 hours. After incubation, light absorbance at wavelength 450 nm (foreground) and 650 nm (background) was measured using a spectrophotometer. For shRNA-mediated proliferation inhibition assays, the shRNA-infected cells were examined for proliferation at 0 or 2 days after plating. To calculate the percent inhibition mediated by each shRNA, the degree of proliferation during the 2-day period for each shRNA was compared to the corresponding degree for a non-specific, control shRNA.

The effects of AKQU and compounds 1–7 on the viability of INS-1 cells were assessed by an Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Seoul, Korea) prior to performing the GSIS assay. In order to determine the non-toxic dose ranges of AKQU and compounds 1–7, the INS-1 cells were seeded at a density of 1.5 × 10⁴ cells/mL in 96-well plates for 24 h and subsequently treated with different concentrations of AKQU and compounds 1–7 for 24 h. Thereafter, 10% (v/v) Ez-Cytox reagent was added to each well and the cells were incubated for 2 h. The optical densities (OD) of each well were measured at 450 nm using a microplate reader (PowerWave XS, Bio-Tek Instruments, Winooski, VT, USA).

Murine macrophages, RAW264.7 cells, in Roswell Park Memorial Institute (RPMI)-1640 medium (supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin) at a concentration of 1 × 10⁵ cells/mL were seeded in a 96-well plate. After 24 h, the different concentrations of tunic fatty acids (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0%) were injected and incubated for another 24 h. All experiments were carried out in triplicate. EZ-Cytox Cell Viability Assay Kit (Daeil Lab service, Chungcheongbuk, Korea) was used to analyze cellular proliferation, as described by Kim et al. [40 (link)]. The cellular proliferation ratio (%) was calculated based on the following formula: $\mathrm{macrophage}\mathrm{proliferation}\mathrm{ratio}(\%)=\frac{\mathrm{the}\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{test}\mathrm{group}}{\mathrm{the}\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{control}\mathrm{group}}\times 100$
Nitric oxide (NO) production was determined to analyze the immunomodulatory activity of fatty acids. Cells were pre-treated, with various concentrations of tunic fatty acids, for 1 h. Anti-inflammation cells were stimulated with LPS (from Escherichia coli O111:B4, Sigma-Aldrich), and immune-enhancement cells were treated without LPS for 24 h. Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) was used for the analysis of the nitric concentration [41 (link)].

Cell viability was determined by using the EZ-CyTox cell viability assay kit (Daeil Lab Service, Seoul, Korea), according to the manufacturer’s instructions. The cell viability was represented by the percentage of control cells.

Huh-7-based NV replicon-bearing cells (HG23 cells) were kindly provided by Dr. Chang40 (link). RAW 264.7 cells were maintained in Dulbecco’s Minimal Essential Medium (DMEM) containing 10% fetal bovine serum (Gibco), 10 mM HEPES, 10 mM sodium bicarbonate, 10 mM nonessential amino acids, and 50 μg/mL gentamicin. Retinol (95144; Sigma-Aldrich) was applied to cells following dilution with ethanol. Prior to in vitro experiments with Retinol, cell viability was confirmed by propidium iodide (PI) staining using a FACScan system (FACSCalibur; BD Biosciences). Prior to all in vitro experiments, cell viability against Retinol was test using EZ-CyTox Cell Viability Assay kit (Daeil Lab Service Co., Ltd, Korea).