Pe conjugated anti il 17a

The following Abs and isotype controls were purchased from e-Biosciences: PE-conjugated anti–IFN-γ and rat IgG2a; PE-conjugated anti–IL-5; PECy5-labeled anti-CD4; anti-CD80 (16-10A1) and anti-CD83 (Michel-17); rat IgG1, IgG2a, and IgG2b; PE-conjugated anti-CD11c (N418); hamster IgG; PE-conjugated anti–IL-17A (eBio17B7); and rat IgG2a. For intracellular staining, cell suspensions from cervical lymph nodes (cLNs) were stimulated with anti-CD3 and anti-CD28 Abs (eBioscience) for 48 h, followed by PMA (50 ng/ml), ionomycin (1 µg/ml), and brefeldin A (10 µg/ml) for 6 h. Cells were washed, incubated with FcR block (1 µg/ml; eBiosciences), and stained for CD4. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin (Sigma), and stained for IL-5, IL-17 and IFN-γ. Cells were analyzed on a FACS Calibur (BD Biosciences) with FCS Express software (DeNovo Software).

Flow cytometry analysis of neutrophils in the small intestine and colon was performed using our previous protocol [42 (link)]. In brief, at the end of each experiment, the mouse intestine was isolated, cleaned from fat and connective tissues, washed thoroughly with PBS to remove all content, opened longitudinally, cut into 0.5 cm pieces (the parts with Peyer’s patches were excluded) and shaken at 220 rpm in 25 mL EDTA solution for 30 min at 37 °C. Samples were centrifuged at 1500× g, and the supernatant was discarded; this process was repeated twice. The tissues were then washed with harvest medium consisting of RPMI 1640, heat-inactivated fetal bovine serum, HGPG (HEPES, L-glutamine, penicillin/streptomycin, and gentamycin) for 5 min before incubation with collagenase (100 U/mL) for 45 min at 37 °C on the shaker followed by centrifugation. All cells were then incubated with anti-CD16/CD32 followed by FITC-conjugated anti-mouse Ly-6G (Gr-1) for neutrophils. For analysis of intracellular cytokines, cells were fixed with Cytofix/Cytoperm solution and stained with PE-conjugated anti-IL-17A (eBioscience, San Diego, CA, USA). A FACSort flow cytometer was used for the acquisition of data, and analysis was made using CELLQuest software v5.2.1 (BD Biosciences, San Diego, CA, USA) [42 (link)].

The phycoerythrin (PE)-conjugated anti-IL-17A and fluorescein isothiocyanate (FITC)-conjugated anti-FoxP3 were purchased from eBioscience (San Diego, CA), and all other antibodies used in flow cytometry were from BD Biosciences (San Jose, CA). For immunostaining of intracellular IL-17A, two samples of freshly heparinized peripheral blood (200 µL each) were incubated for 6 hours with phorbol-12-myristate-13-acetate (PMA, 300 ng/mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 µL/mL, Sigma-Aldrich) in 800 µL of RPMI 1640 medium supplemented with 10% fetal calf serum. Monensin (0.4 µM, BD PharMingen) was added during the first hour of incubation. Then cytofix/cytoperm kit (BD PharMingen), anti-CD3, anti-CD8, anti-IL17, and anti-IFN-γ antibody (mAb) were used in one sample, whereas anti-CD4, anti-CD25, and anti- FoxP3 mAb were used in the other sample according to the manufacturers’ protocols. For Tregs analysis, anti-CD4, anti-CD25, and anti-HLA-DR mAb were added to 200 µL freshly heparinized blood sample, and then the sample was permeabilized and fixed using fix/perm kit (eBioscience) according to the manufacturer’s instructions. After permeabilization, cells were incubated with anti-FoxP3, anti-CTLA-4, and anti-Ki67 mAb. The stained cells were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tritar, USA).