Roswell park memorial institute (rpmi)

Phorbol 12-myristate 13-acetate (PMA), human interferon gamma (IFNγ), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO), sulforaphane (SFN), dimethyl fumarate (DMF), Oltipraz (OTZ) were purchased from Sigma (MERCK). Wogonin (WG) and anti-GAPDH antibody were obtained from Millipore (MERCK). Dual Luciferase (Firefly-Renilla) Assay System was purchased from BPS Bioscience. RPMI (Roswell Park Memorial Institute) 1640 culture medium, phosphate-buffered saline (PBS), fetal bovine serum, and human GM-CSF were obtained from Eurobio Scientific. Tryptic soy broth and tryptic soy agar were from Conda laboratories (Dutscher). Lipofectamine^® LTX & PLUS^™ Reagent, Live/Dead^™ BacLight^™ bacterial viability kit, CellROX^™ Green Oxidative Stress Reagent, Maxima First Strand cDNA synthesis kit, and Trizol were obtained from ThermoFisher scientific. Fluoromount-G^™ (EMS) was purchased from Euromedex. DC Protein Assay Reagents and iTaq SYBR green supermix were purchased from Bio-Rad. Anti-Lamin A/C antibody was purchased from Abcam. Anti-Nrf2 antibody was obtained from ProteinTech. IRDye^® 680CW goat anti-mouse IgG, and IRDye^® 800CW Goat anti-Rabbit IgG secondary antibodies were purchased from LI-COR^® Biosciences.

Pancreatic islets were isolated from rats by collagenase P (Roche Diagnostics, Milano, Italy) perfusion [28] (link) and purified by continuous-density Ficoll gradient. Briefly, the pancreas was distended by bile duct injection of 15 mL 4°C-cold 1 mg/mL collagenase P (Roche Diagnostics) diluted in HEPES-buffered Hank's balanced salt solution (HBSS) (Sigma-Aldrich), and then it was excised and minced. Islets were digested at 37°C for 20 min under constant agitation. Islets were separated from exocrin tissue by centrifugation on a Histopaque (Sigma-Aldrich) discontinuous gradient, were removed from the interface of the layers, were washed in HBSS, and finally resuspended into 10 mL of RPMI (Eurobio, Milano, Italy) supplemented with 10% fetal calf serum (FCS) (Eurobio), 1% L-glutamine (Eurobio), 10 mM glucose (Sigma-Aldrich), penicillin (50 U/ml, Eurobio), streptomycin (50 µg/ml, Eurobio), amphoterycin B (0,2 µg/ml, Eurobio) and 1% HEPES buffer (Sigma-Aldrich) in free floating culture flask. Islets were handpicked under an inverted microscope under sterile conditions and purity was assessed by Dithizone staining (Sigma-Aldrich). For each graft, the total islet mass, expressed as the 150 µm diameter islet equivalent (IE) which was calculated based on volumetric assumptions.
Pancreatic islets were incubated at 37°C (95% air and 5% CO₂) for 1–2 days before transplantation.