Mcc950

Human PBMCs were freshly isolated from Natrium Citrate-treated blood, and were then isolated by Ficoll-Hypaque (TBD science, cat#LDS1075) density-gradient centrifugation. Cells were cultivated in RPMI 1640 medium (Hyclone, cat# SH30809101) supplemented with 2 mM L-glutamine, penicillin/streptomycin (Solarbio, cat# P1400), and 10% fetal bovine serum (FBS, Hyclone, cat# SH30809.01) in Teflon bags and allowed to rest for 24 h prior to stimulation. All incubations were performed at 37℃ in humidified air with 5% CO₂. PBMCs were stimulated with MSU (100ng/mL) and TLR1/2 stimulator Pam3Cys (10 μg/mL) with or without MCC950 (Topscience, cat#T3701, 5 μM) for 6 h, supernatant and cells were collected for ELISA and RT-PCR. PBMC were cultured with or without recombinant human IL-1β (Novoprotein, cat# 0331537, 5 ng/mL), cells were collected at different time points for RT-PCR.

The HLEC line (FHL124) was kindly provided by Dr. J.R. Reddan (Oakland University). The cell line was maintained in HG Dulbecco's modified Eagle’s medium (DMEM; 25.0 mM glucose; Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime Biotechnology, China) in a humidified atmosphere at 37°C with 5% CO₂. The cell culture medium was changed every 2 days and passaged at 80% to 90% confluence. The medium was replaced by normal-glucose DMEM (5.5 mM glucose; Gibco) with 2% FBS before HG stimulation. In the normal glucose (NG) group, HLEC medium contained 5.5 mM glucose, while in the HG groups, additional glucose (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA) was added to bring the final concentration of glucose to 25 to 150 mM. The same gradient concentrations of mannitol (Aladdin, China) were added into the media as osmotic control groups.
MCC950 (MCE, USA), an NLRP3 inflammasome inhibitor, was dissolved in normal-glucose DMEM at a concentration of 1 mM as a storage solution. SRT1720 (Topscience, China), a selective SIRT1 activator, stock solution was prepared in DMSO and at a concentration of 10 mM. The final working concentrations of MCC950 and SRT1720 were 1 µM and 0.5 µM, respectively. The above two compounds were added 1 hour before HG stress.