Pe conjugated anti mouse il 17a

Mice were anesthetized via i.p. injections of ketamine-xylazine (ketamine, 100 mg/kg body weight; xylazine, 10 mg/kg body weight) and perfused with Hanks’ balanced salt solution (HBSS) containing EDTA for blood removal. The small intestines of the mice were longitudinally opened, washed with PBS, and shaken in HBSS containing 2 mM EDTA for 70 s. The intestines were shredded and shaken in RPMI 1640 containing 4% FBS, 0.2 mg/ml collagenase type 1 (Wako), 0.4 mg/ml dispase 1 (Wako), and 10 μg/ml DNase I (Nippon Gene, Japan) for 30 min at 37°C. Cells in the digested tissue were passed through a cell strainer, and single-cell suspensions were pretreated with brefeldin A (100 ng/ml) for 1 h. The cells were then stained with allophycocyanin-conjugated anti-mouse CD3ε (Becton, Dickinson [BD], NJ, USA), Brilliant Violet 421 (BV-421)-conjugated anti-mouse CD4 (BD), phycoerythrin (PE)-conjugated anti-mouse IL-17A (BD), PE-conjugated anti-mouse IL-17F (BD), and fluorescein isothiocyanate-conjugated anti-mouse CD4 (BD) antibodies. These cells were analyzed using a FACSVerse flow cytometer (BD).

Single-cell suspensions of lymphocyte were harvested from spleen and liver of mice. Prior to intracellular cytokine staining, cells were stimulated with PMA and ionomycin (BD Bioscience) in the presence of brefeldin A (BD Bioscience) for 5h. Cells were collected, washed by PBS, and stained with APC-conjugated anti-mouse CD4 antibody in the presence of FcR-Block (BD Bioscience) in dark for 30 min. After the wash, cells were fixed by CytoFix/Cyto Perm buffer (BD Bioscience) and stained with PE-conjugated anti-mouse IL-17A (BD Bioscience) anti-body or isotype control antibody for 30 min. Data were obtained on a FACS Calibur (BD Bioscience) and analyzed using FlowJo 7.6 software.

For Treg detection, cells were first stained with FITC-conjugated anti-mouse CD45 (BD, United States), PE-CY-7-conjugated anti-mouse CD4 (BD, United States), and APC-conjugated anti-mouse CD25 (BD, United States). For Th17 cell detection, cells were stimulated with Cell Stimulation Cocktail in culture medium for 4 h before surface staining using FITC-conjugated anti-mouse CD45 and PE-CY-7-conjugated anti-mouse CD4. After surface staining, cells were fixed and permeabilized with fixation/permeabilization buffer, followed by staining with PE-conjugated anti-mouse Foxp3 (eBIOSCIENCE, United States) for Tregs and PE-conjugated anti-mouse IL-17A (BD, United States) for Th17 cells. For dendritic cells detection, cells were stained with FITC-conjugated anti-mouse CD11c (BD, United States). Cells were analyzed on a FACS flow cytometer (CytoFLEX, Beckman, United States and BD Biosciences, United States).

Anti-mouse CD3e (#553057) and anti-mouse CD28 (#553295), recombinant mouse IL-2 (#575404) and IL-12 (#577004), and FITC-conjugated anti-mouse CD4 (#100406), PerCP-conjugated anti-mouse CD8a (#100732), PE-conjugated anti-mouse/human CD44 (#103008), APC-conjugated anti-mouse CD62L (#104412), PE/Cyanine7-conjugated anti-mouse IFN-γ (#505826), APC-conjugated anti-mouse IL-4 (#504106), APC-conjugated anti-mouse IL-17A (#506916), PE-conjugated anti-mouse IL-17A (#506904), and Alexa Fluor 647-conjugated anti-mouse Foxp3 (#126408) antibodies were purchased from the BD Biosciences and BioLegend (both San Diego, CA, USA). Anti-insulin (#4590s), anti-STAT1 (#14994S), and anti-Phospho-STAT1 (Tyr701) (#9167S) antibodies were obtained from the Cell Signaling Technology (Danvers, MA, USA). Anti-MBD2 (#ab188474) antibody was got from Abcam (Cambridge, MA, USA). The anti-Lamin B1 (#12987-1-AP) antibody was obtained from Proteintech (Wuhan, China).