Murine PMN and Treg Coculture Protocol

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.