Anti il 17a tc11 18h10

Draining lymph nodes (dLNs) and spleen were triturated and dispersed through a 40 μm strainer and then washed with PBS. After washing, single cell suspension was prepared for flow cytometry analysis [6 (link),33 (link)]. For surface staining, cells were stained by using anti-mouse CD3 (145-2C11, BD Bioscience, San Jose, CA, USA), CD4 (GK1.5, BD Bioscience), CD8 (53-6.7, BD Bioscience), CD11b (M1/70, BD Bioscience), CD11c (N418, BD Bioscience), and Gr-1 (RB6-BC5, BD Bioscience). For intracellular cytokine staining, cells were incubated for 4 hours with PMA (200 ng/mL, Merck, NJ, USA) and ionomycin (500 mg/mL, Merck) in the presence of Golgistop (BD Bioscience). After stimulation, cells were surface stained as described above. Then, cells were re-suspended with 250 μL fixation solution (BD Bioscience) and washed by permeabilization buffer (BD Bioscience). Twenty minutes later, cells were stained with intracellular antibodies, i.e., anti-IFN-γ (XMG1.2, eBioscience, San Diego, CA, USA), anti-IL-17A (TC11-18H10, BD Bioscience), anti-GM-CSF (MPI-22E9, Biolegend, San Diego, CA, USA), and anti-IL-10 (JES5-16E3, Biolegend) antibodies. For Ki-67 (SoIA15, eBioscience) staining, cells were fixed and permeabilized by transcription factor staining buffer set (eBioscience) for nuclear protein staining. Results were analyzed by FlowJo (Tree star Inc., Oakland, CA, USA).

Colon cells were stained with Fc Block (Clone 2.4G2) followed by staining with fluorescently labelled antibodies in IEC buffer (PBS with 5% FBS and 2 mM EDTA to reduce cell clumping) for IECs or 2% FBS in PBS for T cells on ice in 1.5 ml microcentrifuge tubes. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Bioscience). Samples were acquired on an Attune NxT flow cytometer (Life Technologies) and analysed with FlowJo software. Cells were sorted on either a BD ARIA II or S6 Enceladus (BD Biosciences). The following antibodies/reagents were used: anti-CD45 (30-F11; Biolegend), anti-EPCAM1 (G8.8; ThermoFisher), anti-FABP2 (polyclonal; R&D/Fisher), anti-goat IgG (ThermoFisher), anti-LY6G (1A8; BioLegend), anti-MHCII (M5/114.15.2; Fisher), Live/Dead Fixable Near-IR dead cell dye (ThermoFisher), anti-CD4 (RM4-5; BioLegend), anti-TCRβ (Η57−597; ThermoFisher), anti-CD44 (IM7; BioLegend), anti-CD45.1 (A20; ThermoFisher), anti-CD45.2 (104; BD Biosciences), anti-IL-17A (TC11-18H10; BD Biosciences), anti-IFNγ (XMG1.2; ThermoFisher), and anti-IL-22 (1H8PWSR; ThermoFisher).