Cell counting kit 8 reagent

KO and HeLa cells were treated with MβCD at 5, 8, 10 and 12 mM for 24 h, and cells without treatment were set as the control. Then, cells were incubated with cell counting kit-8 (CCK8) reagent (Sigma) for 1 h at 37 °C. The peak absorption of each well at 450 nm was then measured using an enzyme marker, and the data were analyzed.

MLR assay was used to evaluate GM-BM-induced proliferation of allogeneic CD4⁺ T cells. Mock-, uvi-ECTV- or ECTV-infected GM-BM were harvested 24 h after culture with or without LPS. Allogeneic CD4⁺ T cells were obtained by negative selection from spleens of uninfected C57BL/6 mice using mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotec) with magnetic cell separation. The purity of CD4⁺ T cells was assessed by flow cytometer and was greater than 90%. Prior to co-culture with T cells, GM-BM were treated with 100 μg/ml of mitomycin C (Sigma-Aldrich) at 37°C for 30 min. Graded dilutions of GM-BM were added to 1 x10⁵ allogeneic CD4⁺ T cell in triplicate in 100 μl of complete medium to flat-bottomed 96-well plate (DCs:T cells = 1:5 and 1:10). GM-BM were co-cultured with allogeneic T cells for 5 days and then 10 μl of Cell Counting Kit-8 (CCK8) reagent (Sigma-Aldrich) was added to each well. After 2 h of incubation the conversion of WST-8 to water-soluble formazan was measured at 450 nm using Epoch Microplate Spectrophotometer. T cell proliferation in each studied group of GM-BM was calculated in relation to values obtained in mock-infected cells, considered as 100%.

Stable cell lines were constructed using the ATF3 lentivirus. The cells were plated into 96-well plates at 1 × 10³ cells/well. Cell viability was measured at 1, 2, 3, and 4 days using Cell Counting Kit-8 (CCK-8) reagent (96992; Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Optical density (OD) was measured at 450 nm using an ELx800 Absorbance Microplate Reader (BioTek, Winooski, VT, USA).