Anti ccr6 29 2l17

Cells were stained with Fixable Viability Dye (eBioscience, San Diego, CA, USA) and the following antibodies: anti-CD45 (30-F11, BioLegend, London, UK), anti-CD4 (RM4-5, BioLegend), anti-CCR6 (29-2L17, BioLegend), anti-IL-7Rα (A7R34, Invitrogen, Waltham, MA, USA), Mouse Hematopoietic Lineage Cocktail (eBioscience): anti-CD3 (17A2), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-TER-119 (TER-119), anti-Gr-1 (RB6-8C5). Intracellular staining was performed using an eBioscience Foxp3/Transcription Factor Staining Buffer Set and the following antibodies: anti-RORγt (B2D, eBioscience), anti-GATA3 (16E10A23, BioLegend). Flow cytometric analysis was performed using FACSAria III flow cytometer (BD Bioscience) and FlowJo software (Tree Star). Cell sorting of splenic ILC3 and CD4⁺ T cells was performed using anti-CD4 (GK1.5; eBioscience) and Hematopoietic Lineage Cocktail (anti-CD3, anti-CD19, anti-CD11c) on FACSAria (BD Bioscience). ILC3 were identified as Lineage⁻CD4⁺ cells, CD4⁺ T cells were identified as Lineage⁺CD4⁺ cells. Purity was more than 90%.

Single cell suspensions obtained from the intestinal mucosa or MLNs were stained and analyzed on FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo software v10.8.2 (TreeStar, Ashland, OR, USA). The following antibodies were used: anti-B220 (RA3-6B2; BioLegend), anti-CD38 (90; BioLegend), anti-CD45 (30-F11; BioLegend), anti-GL7 (GL7; BioLegend), anti-CD138 (281-2; BioLegend), anti-CD4 (GK1.5; BioLegend), anti-CXCR5 (L138D7, BioLegend), anti-PD-1 (29F.1A12, BioLegend), anti-Siglec-F (E50-2440, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD11c (N418, BioLegend), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-IgE (RME-1, BioLegend), anti-CD103 (W19396D, BioLegend), anti-CD11b (M1/70, BioLegend), anti-IL-7Rα (A7R34, BioLegend), anti-CCR6 (29-2L17, BioLegend), anti-TCRβ (H57-597, BioLegend), and anti-TCRγδ (UC7-13D5, BioLegend). Dead cells were gated out using a Zombie NIR Fixable Viability Kit (BioLegend). Before staining, Fc receptors were blocked with an anti-CD16/32 antibody (2.4G2, BioLegend). Negative controls consisted of isotype-matched, directly conjugated, nonspecific antibodies (BD Biosciences). Intracellular staining was performed using anti-Foxp3 antibody (FJK-16s; Thermo Fisher Scientific) and anti-RORγt antibody (Q31-378; BD Biosciences), as described previously [29 (link)].