Cd62l pe vio770

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.