*4.1. Animals*

IFN-γ gene-disrupted (knockout (KO)) mice were generated and established as described previously [37] and backcrossed to C57BL/6 mice for more than eight generations. Wild-type (WT) C57BL/6 mice, purchased from CLEA Japan (Tokyo, Japan), were used as controls. Male or female mice at 7 to 10 weeks of age were used in the experiments. Food and water were available ad libitum. All mice were kept under specific pathogen-free conditions in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine (Sendai, Japan). All experimental protocols described in the present study were approved by the Ethics Review Committee for Animal Experimentation of Tohoku University (2016MdA-279-3, 19 July 2016; 2016MdLMo-138-3, 7 July 2016). All experiments were performed under anesthesia, and all efforts were made to minimize suffering of the animals.

#### *4.2. Wound Creation and Tissue Collection*

All handling of the animals was performed under anesthesia induced by an intraperitoneal injection of 40 mg/kg sodium pentobarbital (Somnopentyl, Kyoritsu Seiyaku Corporation, Tokyo, Japan) and sustained by inhalation anesthesia of isoflurane (Isoflurane, Mairan Pharma, Osaka, Japan). The dorsal hair was shaved to fully expose the n skin, which was then rinsed with 70% ethanol. Four full-thickness wounds extending to the panniculus carnosus were created using a 6 mm diameter biopsy punch (Biopsy Punch, Kai industries Co., Ltd., Gifu, Japan) under sterile conditions. The injured areas were covered with a polyurethane film (Tegaderm Transparent Dressing, 3M Health Care, St. Paul, MN, USA) and an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) as an occlusive dressing. The day on which the wounds were made was designated as Day 0. At various time points, mice were sacrificed, and the wound tissue was collected by excising a 1 cm square section of skin using scissors and a surgical knife.

#### *4.3. Administration of Anti-Gr-1 Antibody and the E*ff*ect of Neutrophil Depletion Induced by This Means*

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).

#### *4.4. Measurement of the Wound Area*

Morphometric analysis was performed on digital images obtained using a digital camera (CX4; Ricoh, Tokyo, Japan). After the wounds were created, photographs were taken of each wound before dressing. At various time points, the polyurethane films were gently removed from the experimental mice, and the wounds were photographed. Each wound area was quantified by tracing its margin and calculating the pixel area using AxioVision imaging software Release 4.6 (Carl Zeiss Micro Imaging Japan, Tokyo, Japan). Percentage of wound closure was calculated using the following formula: % wound closure = (1 − wound area at the indicated time point/wound area on Day 0) × 100.
