Fitc labeled anti mouse cd4 antibody

We counted Th2 andTh17 cells in intestinal mesenteric tissue using a flow cytometry apparatus (BD FACS Melody, USA). We performed Th2 and Th17 double staining using FITC-labeled anti-mouse CD4 antibody (BioLegend, San Diego, CA, USA) and PerCP-labeled anti-mouse IL-4 for Th2 (BioLegend) and FITC-labeled anti-mouse CD4 antibody and PE-labeled anti-mouse IL-17 (BioLegend) for Th17 [30 ].

Mononuclear cells were isolated from mesenteric lymph nodes (mLNs) and spleen. The cells isolated from the spleen samples were then subjected to RBC lysis using RBC lysis buffer (Biolegend, San Diego, CA, USA) to obtain the lymphocytes. The cells were labeled with the Fixable Viability Dye eF780 (eBioscience, San Diego, CA, USA) to identify and exclude the non-viable cells from further analysis selectively. The cells were then subjected to FITC-labeled anti-mouse CD4 antibody (Biolegend, San Diego, CA, USA), PE-Cy7 labeled anti-mouse CD8a (Tonbo Bioscience, San Diego, CA, USA), and APC labeled Nrp1 (R&D SYSTEMS, Minneapolis, MN, USA) for surface staining. After fixation and permeabilization, the cells were stained with PE-labeled Foxp3 Monoclonal antibody (FJK-16s) (eBioscience, San Diego, CA, USA) and APC-labeled Rorgt (eBioscience, San Diego, CA, USA). The stained cells were then analyzed using a BD FACSVerse^TM flow cytometer (BD Bioscience, San Jose, CA, USA). The data analysis was conducted using Flow Jo 10.0 (Three Star, Ashland, OR, USA).