Click it plus edu assay kit

Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's protocol. ESCC cells were seeded in 96-well plates at a density of 1 × 10⁴/mL. After incubation for 0, 24, 48, and 72 h, 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well and the absorbance at 450 nm was measured using a microplate reader after incubation at 37°C for 2 h. The experiments were repeated in triplicate, independently [37 (link)].
An ethynyl deoxyuridine (EdU) was detected using Click-iT Plus EdU Assay Kit (ThermoFisher). Treated cells were observed by fluorescence photo-microscopy with results quantified by counting at least six random fields [38 (link)].

Shenfu injection was obtained from China Resources Sanjiu (Ya’an) Pharmaceutical Co. (Shenzhen, China). Lipopolysaccharide (LPS) was from Sigma-Aldrich (St. Louis, MO). Antibodies (p44/42 MAPK(Erk1/2)(3A7)mouse mAb, phospho-p44/42 MAPK(Erk1/2)(thr202/Tyr204), p38, p-p38) used for western blotting (WB) were from Cell Signalling Technology (Danvers, MA). Claudin-4 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Enzyme-linked immunosorbent assays (ELISA) kits for cytokines were purchased from Abcam (Cambridge, MA). TUNEL Apoptosis Assay Kit was obtained from Roche (Basel, Switzerland). Click-iT Plus EdU Assay Kit was from Thermo Fisher Scientific (Waltham, MA).