To obtain single-cell suspension from dorsal skin, samples were removed from the subcutaneous fat and mucosal tissue with the scalpel and spread into a Petri dish. The samples were then incubated in 2.4 U/mL dispase II overnight at 4 °C and then immersed in DMEM containing 50% (v:v) FBS to inactivate the dispase II. Gently scrape off the epidermal layer and add 5 mL of 0.25% EDTA-free trypsin (Thermo Fisher, 15050057) to the tube to obtain a single cell suspension, after digestion at 37 °C for 20 min. Finally, neutralized with 5 mL DMEM medium (GbicoTM, LS11995065). Single-cell suspension of epidermis cells was made followed by mechanical dissociation with a gentle MACS dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany), and filtered sequentially through 70 μm cell strainers (BD Bioscience, 352350), and cells were washed once with PBS.
To obtain single-cell suspension from lymph nodes, samples were ground in 40 μm cell strainers (BD Bioscience, 352340) with 5 mL PBS solution, and then filtered with 70 μm cell strainers (BD Bioscience, 352350). Cells were washed once with PBS.
For surface staining, the cells were stained with appropriate antibodies against surface antigens in PBS on ice for 30 min. The cellular viability was assessed by staining with 7-amino actinomycin D (7-AAD) (BioLegend, 420404; 0.5 μg/mL) to exclude dead cells. For the analysis of IL-4、IFN-γ and IL-17A production, in vitro re-stimulation, and intracellular staining, single-cell suspensions were incubated for 4 h at 37 °C with PMA (Sigma-Aldrich, p1585; 200 ng/mL), brefeldin A (BioLegend, 420601; 5 μg/mL), and ionomycin (Abcam, ab120116; 1 μg/mL). The cells were then washed and stained with the fixable viability stain 620 (FVS 620; BD-Biosciences, 564996) for 10 min. After performing surface staining as described above, cells were fixed with 4% paraformaldehyde and permeabilized with PBS supplemented with 0.1% Triton X-100. Intracellular staining with fluorescent-labeled antibodies was per- formed for 30 min in PBS. For flow cytometric analysis, the cells were washed and resuspended in PBS. Flow cytometry was per- formed using the NovoCyte flow cytometer and ACEA NovoExpressTM software (ACEA Biosciences, San Diego, CA, USA) and BD LSRFortessaTM and Flow Jo™ software (BD Biosciences, USA). The single-cell suspensions were stained with the following antibodies: CD3-APC-CY7 (100222), CD4-PerCP/Cy5.5 (100434), CD8-PE-CY7 (100722), IFN-γ-FITC (505806), IL-4-APC (562045), IL-17A- PE (506903), CD11b -FITC (11-0112-82), Ly6G-PE-cy7 (108416), Ly6C-APC/Cy7 (1208026), Siglec-F-Bv421 (E50-2440), CD11c-APC/Cy7 (117324), MHCII-FITC(I-A-I-E, 107606). Antibodies were purchased from eBioscience and BioLegend and used at 1:100 dilution.
To obtain single-cell suspension from lymph nodes, samples were ground in 40 μm cell strainers (BD Bioscience, 352340) with 5 mL PBS solution, and then filtered with 70 μm cell strainers (BD Bioscience, 352350). Cells were washed once with PBS.
For surface staining, the cells were stained with appropriate antibodies against surface antigens in PBS on ice for 30 min. The cellular viability was assessed by staining with 7-amino actinomycin D (7-AAD) (BioLegend, 420404; 0.5 μg/mL) to exclude dead cells. For the analysis of IL-4、IFN-γ and IL-17A production, in vitro re-stimulation, and intracellular staining, single-cell suspensions were incubated for 4 h at 37 °C with PMA (Sigma-Aldrich, p1585; 200 ng/mL), brefeldin A (BioLegend, 420601; 5 μg/mL), and ionomycin (Abcam, ab120116; 1 μg/mL). The cells were then washed and stained with the fixable viability stain 620 (FVS 620; BD-Biosciences, 564996) for 10 min. After performing surface staining as described above, cells were fixed with 4% paraformaldehyde and permeabilized with PBS supplemented with 0.1% Triton X-100. Intracellular staining with fluorescent-labeled antibodies was per- formed for 30 min in PBS. For flow cytometric analysis, the cells were washed and resuspended in PBS. Flow cytometry was per- formed using the NovoCyte flow cytometer and ACEA NovoExpressTM software (ACEA Biosciences, San Diego, CA, USA) and BD LSRFortessaTM and Flow Jo™ software (BD Biosciences, USA). The single-cell suspensions were stained with the following antibodies: CD3-APC-CY7 (100222), CD4-PerCP/Cy5.5 (100434), CD8-PE-CY7 (100722), IFN-γ-FITC (505806), IL-4-APC (562045), IL-17A- PE (506903), CD11b -FITC (11-0112-82), Ly6G-PE-cy7 (108416), Ly6C-APC/Cy7 (1208026), Siglec-F-Bv421 (E50-2440), CD11c-APC/Cy7 (117324), MHCII-FITC(I-A-I-E, 107606). Antibodies were purchased from eBioscience and BioLegend and used at 1:100 dilution.
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