Ldh assay kit

The cytotoxicity effects of different glucose concentrations were measured by colorimetric method using an LDH assay kit (Elabscience^® Biotechnology). PDLF cells were seeded in 96-well plates (Thermo Fisher Scientific) at 5 × 10⁴ cells per well in 200 µL of complete culture medium and incubated at (37 °C, 100% humidity, 95% air, and 5% CO₂) upon confluency. Next, culture media containing three different glucose concentrations (5.5, 25, and 50 mM) with or without 1 µg/mL of LPS were added to each appropriate experimental well. After 24 and 48 h, two independent experiments in triplicate wells were carried out following the manufacturer’s protocol. The optical densities of each well were assessed at 450 nm using BioTek^® Synergy™ HT Microplate Reader (BioTek^®).

Pyruvate, Compound C, GW6471, forskolin, IBMX, 8-Bromo-cAMP, H89, ESI-09, 666-15 and MTT were purchased from Merck KGaA (Darmstadt, Germany). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin for cell culture were obtained from Thermo Fisher (Waltham, MA, USA). The kits for RNA extractive, reverse transcription (RT) and quantitative PCR (qPCR) were obtained from Takara Bio Inc (Dalian, China). Protein extraction kits and BCA assay kits were purchased from Bio-Rad Laboratories (Hercules, USA). Human and mouse FGF21 ELISA kits, cAMP assay kits, the ALT activity assay kit, the AST activity assay kit and the LDH assay kit were obtained from Elabscience (Wuhan, China). Pyruvate assay kits were obtained from Abcam (Boston, MA, USA). Anti-cAMP response element-binding protein (CREB) and anti-phospho-CREB (Ser133) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). H&E staining kits were obtained from Beyotime Biotechnology (Beijing, China).