Apc cy7 anti ly6g mab clone 1a8

The cells obtained from the wounded tissues were incubated with Anti-Mouse CD16/CD32 (clone 2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) on ice for 15 min in PBS that contained 1% FCS and 0.1% sodium azide. The cells were stained with Pacific blue-anti-CD45 monoclonal antibody (mAb) (clone 30-F11, BioLegend, San Diego, CA, USA), APC-anti-CD11b mAb (clone M1/70, BioLegend, San Diego, CA, USA), APC/Cy7-anti-Ly6G mAb (clone 1A8, BioLegend, San Diego, CA, USA), and FITC-anti-F4/80 mAb (clone BM8, BioLegend, San Diego, CA, USA). Isotype-matched irrelevant IgG (Pacific: blue Rat IgG2b, κ Isotype Ctrl mAb, APC Rat IgG2b, κ Isotype Ctrl, APC/Cy7 Rat IgG2a, κ Isotype Ctrl, and FITC Rat IgG2a, κ Isotype Ctrl, BioLegend, San Diego, CA, USA) was used for control staining. The stained cells were analyzed using a BD FACS CantoTM II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FACS Diva software. Neutrophils and macrophages were identified as CD45⁺CD11b⁺Ly6G⁺ cells and CD45⁺CD11b⁺F4/80⁺ cells, respectively. The number of neutrophils and macrophages was estimated by multiplying the total leukocyte number by the proportion of each fraction.

Anti-Gr-1 monoclonal antibody was purified from hybridoma culture supernatants (clones RB6-8C5) using a protein G column kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). To neutralize the biological activity of neutrophils, mice were injected intraperitoneally with 400 µg of mAb on Days 5 and 7 after wounding. Rat IgG (ICN Pharmaceuticals, Aurora, OH, USA) was used as a control antibody. Immediately prior to injection and on Days 1, 2 and 5 after injection, mouse blood was collected via the tail vein and reacted with 0.83% ammonium chloride and Tris-HCl (pH 7.2), then washed three times with 1% FCS RPMI 1640 medium, yielding the blood cells used in our flow cytometric analysis. These blood cells were stained with PE-CD11b (BioLegend, San Diego, CA, USA) and APC/Cy7-anti-Ly6G mAb (clone 1A8; BioLegend). Isotype-matched irrelevant IgG was used for control staining. The stained cells were analyzed using a BD FACS Canto II flow cytometer (BD Bioscience, San Jose, CA, USA).