Apc conjugated anti cd11c

EG7 cells were injected subcutaneously. Eighteen hours later, to detect DCs in the skin, skin tissues were excised, cut into small pieces and digested with DNase I (0.1 mg/ml; Sigma) and collagenase Type IV (4 mg/ml; Gibco) for 2 h at 37 °C. The resulting cell suspensions were centrifuged, washed in PBS and passed through a 70-μm nylon mesh filter (BD Biosciences) to obtain single-cell suspensions. The single-cell suspensions were stained with PerCP-Cy^TM5.5-conjugated anti-CD45, PE-conjugated anti-MHC-II, APC-conjugated anti-CD11c (1:100; BD Biosciences) and FITC-conjugated anti-IFN-β (1:100; PBL Assay Science) antibodies for a flow cytometry assay.

For flow cytometry analysis, cell suspensions were incubated in FACS staining buffer (PBS containing 1% BSA) and subsequently stained for 30 min at 4 °C with an optimized concentration of antibodies (BD Bioscience, Franklin Lakes, NJ, USA): PE-conjugated anti-CD3, PerCPCy5.5-conjugated anti-CD8, PE Cy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD45, APC-conjugated anti-CD11c and PE-conjugated anti-SiglecF to determine cell types in the BAL. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and the data were analyzed using the FlowJo software (v 7.6.5; Ashland, OR, USA). Based on surface marker expression, six different cell types were identified: CD45⁺ (total leukocytes), CD45⁺SiglecF⁺CD11c^low (eosinophils), CD45⁺SiglecF⁺CD11c^high (alveolar macrophages), CD45⁺CD3⁺ (total T cells), CD4 T cells (CD45⁺CD3⁺CD4⁺) and CD8 T cells (CD45⁺CD3⁺CD8⁺).

Cells were washed with PBS twice and incubated in a 10% FcR-blocking solution (Miltenyi Biotec Bergisch Gladbach, Germany) for 30 minutes at 4°C to block the nonspecific binding to FcR. Then, cells were stained with PE- or APC-conjugated antibodies. Control staining was performed simultaneously using isotype-matched, irrelevant antibodies also directly conjugated to PE or APC. The fluorescence intensity of the cells was analyzed by flow cytometry (FACS Calibur; Becton Dickinson, United States). The antibodies used are PE-conjugated anti-CD80 and HLA-DR (BD Biosciences, United States) and APC-conjugated anti-CD11c (BD Biosciences, United States).

Immature BMDCs were pre-incubated with epinephrine, propranolol followed by epinephrine or vehicle control and activated with LPS for 24 hrs. After maturation cells were harvested and RALDH activity in individual cells was measured using Aldefluor staining kit (StemCell Technologies, Grenoble, Fr), according to the manufacture's protocol with modifications. Briefly, cells were suspended at 10⁶ cells/ml in Aldefluor assay buffer containing activated Aldefluor reagent (1.5 µM) with or without the RALDH inhibitor diethylaminobenzaldehyde (DEAB, 15 µM) were incubated for 40 min at 37°C. The cells were subsequently stained with APC conjugated anti-CD11c (BD Biosciences) in ice-cold Aldefluor assay buffer. Aldefluor reactive cells were detected using a FACSCalibur flow cytometer. Cell viability was determined by flow cytometry with 7AAD exclusion.

BAL were collected by cannulating the trachea with a 22-gauge cannula, then washing the lungs with 3 x 800μL of cold, sterile PBS. The total volume of the recovered lavage after 3 washes was ~2mL. Cells were initially surface stained with anti-CD16/32 (BD Bioscience) in 0.5% BSA/PBS solution to block non-specific AB interaction with Fc receptors. Cells were then surface-stained with different combinations of PE-conjugated anti-Siglec-F, PE-Cy7-conjugated anti-F4/80, APC-conjugated anti-CD11c, APC-Cy7 anti-CD11b, FITC-conjugated anti-Gr1, PE-Cy5.5-conjugated anti-CD115 (All from BD Biosciences). For NP staining, cells were fixed and permeabilized using BD CytoFix/CytoPerm (BD #554714) before intracellular staining with FITC-conjugated anti-NP (Abcam #ab20343). Flow cytometry was performed using BD LSR II (BD Biosciences) with FACSDiva Software Version 6.1.2 (BD Biosciences). Analysis was performed using FlowJo Software Version 10.0.6 (Tree Star).

BALF samples were collected by making an incision in the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemacytometer.
For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with PE-Cy7-conjugated anti-CD11c (BD Biosciences), APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated Ly6B (Abcom), PCP-Cy5-conjugated anti-Ly6C (eBiosciences) and PE-Cy7 conjugated anti-Ly6G mAb (BD Biosciences) for myeloid cell analysis. APC-conjugated anti-CD11c, APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated anti-MHC II (BD Biosciences) and PCP-Cy5-conjugated anti-F4/80 (eBiosciences) were used for DC analysis. PCP-Cy5-conjugated anti-CD3 (Biolegend), APC-conjugated anti-CD4 (BD Biosciences) and APC-Cy7-conjugated anti-CD8 mAb (BD Biosciences) were used for T cell analysis. Splenocytes and BALF cells were stained with Tetramer Alexa 647-Labeled H-2D(b)/PA₂₂₄(SSLENFRAYV) and BV421-Labeled H-2D(b)/NP₃₆₆ (ASNENMETM), using FITC-conjugated anti-CD3 (BD Biosciences), APC-Cy7-conjugated anti-CD4 (BD Biosciences) and PE-conjugated anti-CD8 mAb (BD Biosciences) for cell surface markers. The stained cells were analyzed on a BD FACSCanto or BD LSRII-green using FlowJo and BD FACSDiva software analysis.