Cell counting kit 8 assay solution

After the cells were incubated for 24 h to 48 h, 10 µL/well of Cell Counting Kit-8 assay solution (Dojindo Laboratories) was added and incubated for 2 h. Absorbance was then measured at 450 nm and 650 nm using a microplate reader. The net difference in absorbance (A450 – A650) was used as a measure of cell viability.

The spleen lymphocytes were isolated from immunized mice at 28 dpi and re-suspended with RPMI-1640 medium containing 10% FBS. The spleen lymphocytes were seeded into a 96-well plate at a density of 2 × 10⁶ cells per well and were then stimulated by adding various concentrations (2.5 × 10⁴, 1 × 10⁵, 4 × 10⁵ HAD₅₀/well) of heat-inactivated ASFV. Concanavalin A, unstimulated cells, and RPMI-1640 medium were set as positive, negative, and blank controls, respectively. The level of cytokines in supernatants in each well was detected after the plate was incubated for 72 h at 37°C. The Cell Counting Kit-8 assay solution (CCK8, Dojindo, Japan) was added and incubated for 4 h at 37°C. Finally, the absorbance of OD₄₅₀ was measured. The calculated formula is as follows: the stimulation index SI = (experimental group OD₄₅₀ - blank control OD₄₅₀) / (negative control OD₄₅₀ - blank control OD₄₅₀).

After the cells were cultured for 24 to 60 h, a volume of 10 µL/well of the Cell Counting Kit-8 assay solution (Dojindo Laboratories, Kumamoto, Japan) was added and the cells were further incubated for 1 h. Absorbance was then measured at 450 nm and 650 nm using a microplate reader. The net difference in absorbance (A450–A650) was used as a measure of metabolic activity. The metabolic activity of the siRNA control-treated cells was considered to be 100%.
After the cells were cultured for 24 to 60 h, an equal amount of the CellTiter-Glo 2.0 Cell Viability Assay reagent (Promega, Madison, WI, USA) was added to each well and incubated for 10 min. Luminescence was measured using a luminometer to determine the amount of ATP, as well as cell proliferation. The amount of ATP in the siRNA control-treated cells was considered to be 100%.