*4.5. Flow Cytometry*

Cells were isolated from the wound tissues as described previously [6]. Thus, the tissues were cut into small pieces and digested at 37 ◦C for 90 min in DMEM containing 10% FBS, 1.2 mg/mL hyaluronidase (Sigma-Aldrich), 2 mg/mL collagenase (Sigma-Aldrich), and 0.2 mg/mL DNase I (Sigma-Aldrich). The cell pellets were resuspended in PBS containing 2% FBS and then incubated with anti-CD16/32 antibody (BioLegend, San Diego, CA, USA) for 5 min to block the Fc γ receptors. To measure inflammatory cell recruitment, the wound cell preparations were stained with phycoerythrin (PE)-conjugated anti-Gr-1, allophycocyanin (APC)-conjugated anti-CD3a, PE/CY7-conjugated anti-CD4, or fluorescein isothiocyanate (FITC)-conjugated anti-CD8a antibody (CiteAb, Bath, UK) at 4 ◦C for 20 min. The lymphocytes were analyzed with FACSDiva (BD, San Jose, CA, USA).
