Golgi stop plug

Dissociated cells from pancreatic lymph nodes were suspended in buffer containing 2% FBS in PBS. To stain for surface antigens, cells were incubated with specific antibodies to F4/80 (BM-8, Biolegend), CD11c (HL3, BD Pharmigen), CD4 (RM4–5, Biolegend), CD45 (30- F11, BD Biosciences), CD8a (53–6.7, eBioscience), CD80 (16-10A1, Biolegend), CD274 (10F.9G2, Biolegend), CD86 (GL-1, Biolegend), and MHC-II (M5/114.15.2, Biolegend) or the appropriate isotype controls for 30 min. For the intracellular staining, cells were stimulated with 100ng/mL PMA (Sigma Aldrich), 500ng/ml Ionomycin (Sigma Aldrich), and Golgi Stop Plug (BD Pharmigen, 1/1000). After stimulation, cells were first stained for surface antigens, then fixed and permeabilized with BD Cytofix/Cytoperm™ (BD Pharmigen), according to the manufacturer’s recommendations. Cells were then incubated with specific antibodies to TNF-α (MP6-XT22, Biolegend), IL-1β (NJTEN3, Thermo Fisher Scientific), IL-17 (TC11-18H10, BD Pharmigen), IFN-γ (XMG12, BD Pharmigen), and FoxP3 (MF23, BD Pharmigen). After staining, the cells were washed, filtered, and analyzed on a FACS Canto II cytometer (BD). Data were analyzed using FlowJo software (Tree Star).

Islets isolated from Alox15^−/− or wildtype littermate were stimulated with a cytokine cocktail containing 25 ng/mL IL-1β (Prospec), 50 ng/mL TNF-α (Prospec), and 100 ng/mL IFN-γ (Prospec) for 6 h. Islets were then removed from the stimulated media, washed, and cocultured with peritoneal macrophages (5 × 10⁵ per well) and naive T cells (1 × 10⁵ per well). Mouse peritoneal macrophages were isolated following euthanasia by injecting ice-cold RPMI into the peritoneal cavity using a 25-gauge needle (Ray and Dittel, 2010 (link)). The injected RPMI was then removed, and the isolated cells were lysed with RBC lysis buffer (eBioscience) to remove red blood cells. NaïveT cells were isolated and purified from spleenof C57BL/6J mice using EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies) and stimulated with anti-CD3 (3 μg/mL, BD Pharmigen) and anti-CD28 (5 μg/mL, BD Pharmigen) in U-bottom 96-well plates for 96 h. Cells were then stimulated with 100 ng/mL PMA (Sigma Aldrich), 500 ng/mL Ionomycin (Sigma Aldrich), and Golgi Stop Plug (BD Pharmigen, 1/1000) for 4 h and then stained for FoxP3, IL-17 and IFN-γ and analyzed by FACS cytometry.