Computational protocol: CD11c depletion severely disrupts Th2 induction and development in vivo

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Protocol publication

[…] After FcR-Block (2.4G2), cell surface markers for several different cell populations were analyzed. Staining for CD11c+ subsets was performed using the following mAb conjugations: CD11c-APC, MHCII-PerCP/Cy5.5, B220-APC/eFluor780, CD11b-PE, and CD8-PE/Cy7. In pLN and spleen, DCs were defined as CD11cHiMHCII+, and in the liver as CD11cHiMHCII+CD11bLo. For egg injection experiments, IL-4-eGFP+ basophils were identified by exclusion of cells expressing CD4-APC, CCR3-PE, CD117-PerCP/Cy5.5, or B220-APC/eFluor780. For infection experiments, FcεR1α-FITC+ basophils were identified after surface staining to exclude cells expressing Siglec-F-PE, CD19-Alexa Fluor 700 or eFluor450, and CD117-PerCP/Cy5.5. Macrophages were identified using F4/80-FITC, CD11b-PE, and CD11c-APC, and eosinophils with Siglec-F-PE and Gr-1-APC. T cells were stained with CD4-APC/eFluor780, CD8-PE/Cy7, CD25-PerCP/Cy5.5, and FoxP3-APC. FoxP3 staining was performed as per manufacturer instructions (eBioscience). B cells were stained with B220-APC/eFluor780, CD21-FITC, and CD23-PE. IL-4-eGFP was measured on LN cells directly ex vivo, with naive mice expressing negligible levels of eGFP (0.5–1.5% of CD4+ T cells). Intracellular cytokine production was measured either directly ex vivo or after restimulation. Intracellular cytokine staining of splenocytes or liver leukocytes ex vivo was set up as follows: cells were rested overnight, stimulated with 10 ng/ml PMA and 1 µg/ml Ionomycin (Sigma-Aldrich) for 2 h, and then stimulated with Golgi Stop (BD) for an additional 3 h. After restimulation, intracellular cytokine staining was set-up as follows: after 72 h, 10 ng/ml murine rIL-2 (Peprotech) was added to pLN cell cultures. After an additional 18 h, cells were stimulated with PMA and ionomycin and treated with GolgiStop. After FcR-Block, cells were stained with CD4-FITC or CD4-APC/eFluor780, fixed with 1% isotonic formaldehyde, permeabilized with BD Perm/Wash buffer (BD), and stained with IL-4-APC, IFN-γ-Alexa Fluor 488, or IFN-γ-APC in BD Perm/Wash buffer. Identification of intracellular cytokine–positive cells was determined using appropriate isotype controls. All Abs for flow cytometry were purchased from BD, eBioscience, or BioLegend. Samples were acquired using a FACS LSR II or FACSCanto II flow cytometer using BD FACSDiva software and analyzed with FlowJo v.8 software (Tree Star, Inc.). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Schistosoma mansoni
Diseases Infection, Schistosomiasis mansoni
Chemicals Interleukin-6