Cell counting kit 8 cck 8

The T24 and 5637 human bladder cancer cells were cultured in DMEM medium and RPMI-1640 medium respectively, containing 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin (Life technology, USA) at 37°C in 5% CO₂ humidified incubator. Cell Counting Kit-8 (CCK8) was obtained from Trans Gen Biotech, China. Transwell plates were from Corning Incorporated, USA. Caspase-3, PARP, NF-κB p65, p-NF-κB p65, PI3K, p-PI3K, AKT, p-AKT (Thr 308, Ser473) were purchased from Cell signaling technology, USA. Perifosine, AKT signaling pathway inhibitor, was provided by Selleck, USA.

Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) (TransGen, Beijing, China) and 5-ethynyl-20-deoxyuridine (Edu) assay kit (Ribobio, Guangzhou, China) China according to the instructions. For CCK-8 assays, cells were incubated in a 96-well plate for 24 h and then transiently transfected with CRISPR-Cas13a. The absorbance was detected at 0, 24, 48, and 72 h after transfection by a microplate reader (Bio-Rad, Hercules, CA, United States). Each test was carried out at least three times.

Cytotoxicity was measured using the Cell Counting Kit-8 (CCK-8) assay (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Marc-145 cells and 293T cells were seeded at a density of 5 × 10³ cells per well in complete medium in 96-well plates. After 12 h of culture, nocodazole was added to each well at specific concentrations and incubated for 48 h at 37 °C. Dimethyl sulfoxide-treated cells were included as controls. After treatment, the medium was removed and changed to 100 µL of PBS containing 10% CCK-8 solution. After incubation for 2 h at 37 °C, cell viability was detected by measuring the absorbance at 450 nm using a microplate reader (Bio-Rad model 680). The above experiment was repeated three times.
After infection with PRRSV-2, Marc-145 cells were incubated in maintenance medium containing 0.08, 0.16, or 0.32 μg/mL nocodazole for 24 h, and then harvested for Western blot analysis. 293T cells were transfected with pEGFP-C1 and GFP-NSP2 plasmids for 6 h. The cells were incubated with different concentrations of nocodazole (0.08, 0.16, and 0.32 μg/mL) for 4 h, and then collected at 24 h after transfection for Western blot analysis. 293T cells were also incubated with 0.32 μg/mL nocodazole for 4 h, and then collected at different time points for Western blot analyses.

The materials used included polycaprolactone (PCL, M_n 10 000 Da, Sigma-Aldrich, U.S), maleimide–poly(ethylene glycol)–poly(ε-caprolactone) (MAL–PEG–PCL, M_n 5000 Da, Xi'an Ruixi Biotech, China), Dulbecco's Modified Eagle Medium (DMEM, Hyclone), fetal bovine serum (FBS, Gibco, Australia), osteoprotegerin (OPG, PeproTech, U.S.), OPG ELISA kit (Cloud-Clone Corp, U.S), soybean phosphatidylcholine (SPC, Shanghai Taiwei Biotech, China), Cell Counting Kit 8 (CCK-8, Beijing Transgen Biotech, China), dexamethasone, ascorbic acid, β-glycerol phosphate (Sigma-Aldrich, U.S.), QuantiChrom™ Calcium Assay Kit (BioAssay Systems, U.S), Hematoxylin and Eosin staining kit (H&E staining, Wuhan Boster Biological Technology, China), Masson's Trichrome Stain Kit (MASSON Staining, Genmed Scientifics. Inc, U.S), and osteocalcin antibody (OCN, Wuhan Boster Biological Technology, China). Male Sprague-Dawley (SD) rats weighing 175 ± 26 g, 4 weeks old, were provided by the Experimental Animal Center of Nanchang University. All research protocols were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Nanchang University.

Lentinan (DX0085) was obtained from Desite^@ (Chengdu, China). Ursolic acid (A1810063) was obtained from Aladdin^@ (Shanghai, China). BCA Protein Assay Kit (DQ111-01) and Cell Counting Kit-8 (CCK-8, FC101-02) were purchased from TransGen Biotech Co., Ltd. (Beijing, China). Live/Dead Cell Staining Kit (C542) and Annexin V-FITC/PI Apoptosis Detection (AD10) were obtained from Dojindo (JPN). Primary antibodies were purchased from Abcam (USA). Fluorescent secondary antibodies and streaming antibodies were purchased from Thermo Fisher Scientific Inc. (USA). Adenosine triphosphate (ATP) assay kit (S0026) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay kit (C1088) were obtained from Beyotime Biotech Co., Ltd. (Shanghai, China). In vivo MAb anti-mouse CD47 (BE0270) was obtained from Bio X Cell (USA). Azoxymethane (AOM, A5486) was purchased from Sigma (USA). Dextran sulfate sodium (DSS, 216011090) was obtained from MP Biomedicals (USA). Granulocyte macrophage colony-stimulating factors (GM-CSF, 415-ML-020/CF) and interleukin-4 (IL-4, 404-ML-010/CF) were purchased from R&D Systems (USA). Enzyme-Linked Immunosorbent Assay (ELISA) kits were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China). All other chemicals were used as obtained unless otherwise specified.