Mouse th1 th2 th17 th22 13plex flowcytomix

One centimeter samples of distal colon were recovered and homogenized in an appropriate volume (to give 50 mg/ml) of Tris–HCl buffer containing protease inhibitors (Sigma-Aldrich) in a Tissue Lyser (Qiagen). The samples were centrifuged for 20 min and the supernatants collected and frozen at −80°C until analysis. Blood samples were obtained from the retro-orbital venous plexus before the mice were killed and centrifuged, and the sera stored at −80°C until analysis. IL-6, IL-10, IFN-γ, TNF-α, IL-5, IL-2, IL-22, IL-1α, IL-13, IL-17, IL-4, IL-27 and IL-12p70 were assayed with a cytometric bead array system (Mouse Th1/Th2/Th17/Th22 13plex Flowcytomix) (eBioscience, San Diego, USA).

Mononuclear cells were isolated from spleens and mesenteric lymphoid nodes (MLN) by gentle extrusion of the tissue through a 50 µm-mesh Nylon cell strainer (BD Biosciences, San Jose, CA, USA) and subsequent lysis of erythrocytes in red blood cell lysing buffer (Sigma-Aldrich) as previously described [52 (link)].
For flow cytometry analysis, aliquots of 10⁶–10⁷ cells per sample were pre-incubated with purified anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) and then labelled with anti-CD3-FITC, anti-CD4-PerCP, anti-Tbet-APC and anti-Gata3-PE (all from eBioscience) according to the manufacturer’s instructions. Intracellular staining was performed with the FoxP3 Staining kit (eBioscience). Stained cells were analysed by flow cytometry (Accuri, Ann Arbor, MI, USA) with CFlowSampler software (BD Accuri, Ann Arbor, MI, USA).
For stimulation experiments, 2 × 10⁵ cells isolated from spleens or MLN were cultured for 48 h (37 °C, 10% CO₂) in DMEM medium in P24 plates pre-coated with anti-CD3/CD28 antibodies (4 µg/mL each; eBioscience) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (cell stimulation cocktail, 1×, ebioscience). Culture supernatant was frozen at −80 °C until processing and cytokine concentration determined by a cytometric bead array system (Mouse Th1/Th2/Th17/Th22 13plex Flowcytomix) (eBioscience) according to the manufacturer’s instructions.

Blood samples were obtained from the retro-orbital venous plexus before the mice were euthanized and centrifuged, and the sera stored at −80°C until analysis. One centimeter samples of distal colon were recovered and homogenized in an appropriate volume of PBS (final concentration of 50 mg/ml) in a Tissue Lyser (Qiagen). IL-6, IL-10, IFN-γ, TNF-α, IL-5, IL-2, IL-22, IL-1α, IL-13, IL-17, IL-4, IL-27, and IL-12p70 were assayed in blood and colon samples with a cytometric bead array system (Mouse Th1/Th2/Th17/Th22 13plex Flowcytomix; eBioscience, San Diego, CA, USA). For cytokine quantification in cell culture supernatants the following ELISA tests were performed according to manufacturer’s instruction: IL-4, IL-5, IFNγ, IL-17, IL-12p70, and IL-10 (MabTech); TGFβ and IL-22 (ebioscience).