Cd62l pe cf594

Lung and nasal tissue mononuclear cell suspensions were prepared by mechanical (chopping with a scalpel) followed by enzymatic disruption of tissue for 1 h at 37°C with Collagenase D (1 mg/ml; Sigma-Aldrich) and DNAse I (20 U/ml; Sigma-Aldrich). Next, lungs or spleens were passed through a 40-mm cell strainer to a obtain single-cell suspension, followed by RBC lysis. The cells were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. To detect cytokines, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 mg/ml) for 4 h at 37°C. The following surface Abs were used: CD45R-PE, CD3-BV421, CD44-BV605 (Biolegend), CD62L-PE-CF594 (BD), CD103-BV786, CD4-APC-eF780, CD69-FITC (eBioscience). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–PerCP-Cy5.5 and IFN-γ-BV650 (eBiosciences). Fluorescence minus one or non-specific isotype Abs were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

Lung tissue was chopped and digested with collagenase-D (1 mg/ml; Roche) and DNase I (10 mg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. To detect cytokines, brefeldin A (5 µg/ml) was added during the digest. Lungs or spleens were passed through a 70-µm cell strainer to a obtain single-cell suspension, followed by RBC lysis with ACK buffer. The cells were incubated with Fcγblock (anti-CD16/CD32 antibody, BD Biosciences) (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. The following surface Abs were used: CD69-FITC, CD3-APC-ef780, MHCII-APC, Ly6C-PerCP-Cy5.5, CD86-FITC, CD11b-APC-ef780, CD38-ef450, F4/80-PE-Cy5, CD49d-PerCP-ef710, CD3-AF700, CD8-APC-ef780, CD44-PE-Cy7, CD4-PE-Cy5 (eBiosciences), CD44-BV605, CD4-BV785, CD103-PE, Ly6G-BV605, CD80-PE-Dazzle594 (BioLegend), CD62L-PE-CF594, SiglecF-PE, CD103-BV786 (BD Biosciences). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 (BD Biosciences). Fluorescence minus one samples were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).