A cell counting kit 8 cck 8

Cells and Virus. Vero E6 cells (ATCC no. 1586) were grown and maintained in minimum Eagle’s medium (Gibco Invitrogen) and supplemented with 10% fetal bovine serum (Gibco Invitrogen) at 37 °C in 5% CO₂. The SARS-CoV-2 strain (nCoV-2019BetaCoV/Wuhan/WIV04/2019) was propagated, stored and titrated [6 (link)]. Cytotoxicity and Antiviral Assays. Cytotoxicity was evaluated in Vero E6 cells using a cell counting kit-8 (CCK8) (Beyotime, China) according to the manufacturer’s instructions. For the antiviral assay, 4.8 × 10⁶ Vero E6 cells were seeded onto 48-well cell-culture Petri dishes and grown overnight. After pretreatment with a gradient of diluted experimental compounds for 1 h at 37 °C, cells were infected with the virus at an MOI of 0.01 for 1 h. After incubation, the inoculum was removed, cells were washed with PBS and culture vessels were replenished with fresh drug-containing medium. At 24 h post infection, total RNA was extracted from the supernatant and qRT-PCR was performed to quantify the virus yield [6 (link)]. Results are representative of n = 6 and are shown as mean ± SEM. EC₅₀ and CC₅₀ for each compound were calculated by a four-parameter nonlinear regression model and were plotted by Excel graph.

Palmitic acid (PA) was purchased from Aladdin (Shanghai, China). Oleic acid (OA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), and stearic acid (SA) were purchased from Sigma (Shanghai, China). Bovine serum albumin (BSA) was purchased from Solarbio (Beijing, China). A Cell Counting Kit-8 (CCK-8) was purchased from Beyotime (Shanghai, China). Anti-Cleaved caspase-3, anti-IRE1α antibody, anti-p-eif2a antibody, and anti-BAX antibody were purchased from Cell Signaling Technology (Hong kong, China). Donkey Anti-Mouse IgG H&L (Alexa Fluor^® 488), Donkey Anti-Rabbit IgG H&L (DyLight^® 550), and anti-BCL-2 antibody were purchased from Abcam (Cambridge, UK). Anti-ATF6 antibody and anti-CHOP antibody were purchased from Affinity bioscience (Jiangsu, China). Anti-actin antibody was purchased from ABclonal (Shanghai, China). Anti-BIP antibody and anti-glucagon antibody was purchased from absin (Shanghai, China). Anti-Insulin antibody and anti-ATF4 antibody were purchased from Huaan (Hangzhou, China). Anti-XBP-1 antibody and HRP labelled goat anti-rabbit IgG were purchased from Wanlei (Shenyang, China). TRIizol reagent was purchased from Ambion (TEXAS, USA). A high-capacity cDNA synthesis kit was purchased from Vazyme (Nanjing, China).