Cell suspensions from SPL, mLN, and cLP were analyzed by flow cytometry. Conventional CD4+ T cells and Tregs were stained with anti-CD3ε and anti-CD4 mAbs (BD Biosciences, San Jose, CA) and intracytoplasmic anti-foxp3 mAbs (eBioscience, San Diego, CA). Neutrophils were stained with Gr-1-FITC, CD11b-PECy7, and Ly6G-AF647 mAbs (eBioscience). Plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) were stained with MHCII-FITC, CD11b-PECy7, PDCA-1-AF647, and CD11c-APCCy7 mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin. The cDC subsets were stained with MHCII-FITC, CD11c-APCCy7, CD11b-PECy7, and CD103-PE mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin (eBioscience). Monocytes and macrophages (MΦ) were stained with Gr-1-FITC, MHCII-PE, Ly6C-PerCP-Cy5.5, CD11b-PECy7, Ly6G-APC, and CD11c-APCCy7 mAbs (eBioscience). Blocking of FcγR binding was performed using mouse and rat serum (Jackson ImmunoResearch, West Grove, PA). Cells were analyzed on a FACS Canto II (BD Biosciences). Data were collected using FACS Diva software (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).
Wurbel M.A., Bras S.L., Ibourk M., Pardo M., McIntire M.G., Coco D., Geha R.S., Fiebiger E, & Snapper S.B. (2014). CCL25/CCR9 Interactions Are Not Essential for Colitis Development but Are Required for Innate Immune Cell Protection from Chronic Experimental Murine Colitis. Inflammatory bowel diseases, 20(7), 1165-1176.
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