Rat igg2a isotype control pe

FITC anti-mouse CD3; PE anti-human/mouse Integrin β7 (Biolegend, San Diego, CA, USA); APC anti-mouse CD45R/B220; anti-Mouse/Rat Foxp3 PE; anti-Mouse CD4 FITC; anti-Mouse CD25 APC; rat IgG2a Isotype Control PE (eBioscience, Carlsbad, CA, USA); Alexa 594 anti-mouse; Alexa 488 anti-rabbit; anti-glucagon; anti-insulin (Innovex Biosciences, Richmond, CA, USA); recombinant mouse MAdCAM-1 Fc chimera (R&D BioSystems, Minneapolis, MN, USA); murine TNF-α (Peprotech, Rocky Hill, NJ, USA); mouse IL-1β, IFNγ, and IL-17 ELISA kits (R&D Biosystems). AS101 and SAS were supplied by M. Albeck, Bar-Ilan University, Ramat-Gan, Israel, and were synthesized at Bar-Ilan University.

Spleens from healthy WT and IL-17 deficient mice were collected in sterile Falcon tubes containing PBS (Sigma) and processed individually to obtain single cell suspensions. Briefly, spleens were mashed through two cell strainers (70μm and 40μm) and rinsed with PBS (Sigma). Suspended cells were transferred to a 50ml Falcon tube and spun at 350xg. RBC were lysed by re-suspending cell pellets in 1X RBC Lysis Buffer (Biolegend). Cell viability was assessed using the trypan blue exclusion test, and cells were counted using a haemocytometer.
Flow cytometric analysis of IL-17 receptor expression was performed using Anti-Mouse CD217 (IL-17 Receptor A) PE (Cat. no 12-7182-80, eBioscience) and Rat IgG2a Isotype Control PE (Cat. no 12-4321-73, eBioscience) on a BD LSRFortessa cytometer. Staining was performed for 20 minutes followed by washing. For the analyses, samples were gated on splenocyte population based on size, as assessed by SSC and FSC. Analysis was performed using FlowJo analysis software.

MF (C₁₉H₁₈O₁₁, FW = 422.34, purity ≥ 95%) was purchased from Nanjing ZeLang Medical Technology Co. Ltd. (Nanjing, China). LPS (Escherichia coli, 0111: B4), D-GalN, and ZnPP IX were obtained from Sigma (St. Louis, MO, USA). ALT and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL, USA). Trizol reagent was purchased from Invitrogen (Grand Island, NY, USA). AMV transcriptase, RNasin, SYBR green PCR Master Mix, and the Dual-Luciferase Reporter Assay kit were obtained from Promega (Madison, WI, USA). The tumor necrosis factor (TNF)-α ELISA kit was obtained from Bender MedSystems (Vienna, Austria). Rabbit HO-1 antibody was obtained from Abcam (Cambridge, MA, UK), rabbit β-actin antibody was purchased from Cell Signaling Technology (Boston, MA, USA), and the rat anti-mouse TLR4/MD-2 complex and rat IgG2a isotype control PE were obtained from eBioscience (San Diego, CA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA).