All anti-mouse antibodies for fluorescence activated cell sorting (FACS) and OneComp eBeads were purchased from eBioscience: Blocking CD16/CD32, for monocytes (CD11c PE-eFluor®610, IgG Isoytpe control PE-eFluor®610, Ly-6C APC, IgG1 K Isoytpe control APC), for neutrophils [Ly-6G(Gr-1) PerCP-Cyanine5.5, IgG2b K isotype PerCP-Cyanine5.5, CD11b PE-Cyanine7, IgG1 K Isoytpe control PE-Cyanine7], for macrophages (F4/80 FITC, IgG1 K isoytpe control FITC, CD11b PE-Cyanine7, IgG1 K Isoytpe control PE-Cyanine7). 7 mm wound with 3 mm surrounding tissue were collected at the given time points.
Wound tissues were then cut into small pieces with scissors and combined with 100 ml of collagenase/dispase (1 mg/ml) with RPMI medium and then incubated for 45 min at 37 • C. The treated tissue/cell suspension was then passed through 18 and 20 gauge needles followed by straining using a 70 μm cell strainer (BD Bioscience). Cells were then washed with RPMI. The percoll gradient method was then used to separate neutrophils and monocytes/macrophages from the cell suspension. Collected cells were washed with RPMI and incubated in 10 % purified CD16/CD32 in FACS buffer (10 μl) for 5 min to block non-specific binding to antibodies. Cells were then collected and re-suspended in 100 μl FACS buffer with 1 μl of each antibody and incubated on ice for 30 min and then followed by FACS analysis.
Wound tissues were then cut into small pieces with scissors and combined with 100 ml of collagenase/dispase (1 mg/ml) with RPMI medium and then incubated for 45 min at 37 • C. The treated tissue/cell suspension was then passed through 18 and 20 gauge needles followed by straining using a 70 μm cell strainer (BD Bioscience). Cells were then washed with RPMI. The percoll gradient method was then used to separate neutrophils and monocytes/macrophages from the cell suspension. Collected cells were washed with RPMI and incubated in 10 % purified CD16/CD32 in FACS buffer (10 μl) for 5 min to block non-specific binding to antibodies. Cells were then collected and re-suspended in 100 μl FACS buffer with 1 μl of each antibody and incubated on ice for 30 min and then followed by FACS analysis.