Ly6g apc vio770

For FACS analysis, healthy and lesional skin (both from corresponding anatomical sites) or blood was taken from mice after induction of experimental EBA. Single cell solutions from and blood were erythrocyte lysed with RB cell lysis buffer (Miltenyi). The skin samples were cut into small pieces and digested with 345 mg/ml liberase (Roche) in RPMI for 30 min/37°C. Single cells were stained for the following surface markers using standard FACS procedures: CD45-VioGreen (clone: 30F11), CD3-VioBlue (clone: 17A2), Ly6C-FITC (clone: 1G7.G10), as well as Ly6G-APC Vio770 (clone: 1A8) and for blood CD19-APC (clone: 6D5), all from Miltenyi. For the subsequent intracellular staining, the cells were fixed in fixation buffer (BioLegend) and permeabilized using the Intracellular Staining Perm Wash Buffer (BioLegend) following the manufacturer’s protocol. Intracellular staining was performed with SYK-PE (clone: 4D10.2). Cells were first gated for scatter (SSC-A/FSC-A) and singlets (FSC-H/FSC-A). The CD45⁺ gates were further analyzed for double-positive staining of SYK with the appropriate cell markers. Measurements were performed at the Miltenyi MacsQuant10, and data were analyzed with the MACSQuantify™ Software (version 2.8).

TCRγδ or NKT cells were isolated from murine spleens using the TCRγ/δ + T Cell Isolation Kit (using BALB/cJ mice) or the NK1.1 + iNKT Cell Isolation Kit (using C57BL/6 J mice), respectively (Miltenyi), following the manufacturer’s protocol. The enrichment of cells was determined by FACS. Isolated TCRγδ cells were 78% positive by staining with CD3-PE/TCRγδ−BV421 (Biolegend), isolated NKT cells had 77% positive staining using CD3-PE/CD49b-APC (Biolegend). Isolated TCRγδ or NKT cells were restimulated for 18 h using 5 μg/ml anti mCD3 (Biolegend, clone 145-2C11). The cultured cells, the supernatant or the combination of both was subsequently co-cultured with freshly isolated murine neutrophils50 (link) in a ratio of 1:4 for additional 4 h. The neutrophils were stained for FACS using CD18-FITC, CD62L-PE-Vio770, Ly6G-APC-Vio770 and CD45-VioGreen (Miltenyi) following standard procedures to evaluate the activation status. Dead cells were excluded using PI. The supernatant of cultured cells was analyzed for cytokine release using the LEGENDplex™ Mouse Proinflammatory Chemokine Panel (Biolegend).

PMNs were isolated from the femurs and tibias of healthy C57BL/6J mice as described in detail elsewhere (16 (link)). Tregs were isolated from the spleen of the same animal using a CD4⁺CD25⁺ Regulatory T Cell Isolation Kit, mouse (Miltenyi) following the manufacturer’s protocol. The enrichment of cells was determined by FACS. In total, 2 × 10⁵ PMNs/100 μl were stimulated with ICs formed by 10 µg/ml mCOL7 and 2 µg/ml rabbit anti-mCOL7 IgG as described elsewhere (22 (link)) for 60 min at 37°C. Isolated Tregs were then cocultured with the IC-stimulated PMNs for an additional 4.5 h in a ratio of 1:4 (5 × 10⁴ Tregs/2 × 10⁵ PMN/200 μl). To evaluate the activation status, cells were stained for flow cytometry analysis using CD18-FITC, CD62L-PE-Vio770, Ly6G-APC-Vio770, and CD45-VioGreen (Miltenyi) following standard procedures. Dead cells were excluded using PI.