Ly6g pe clone 1a8

To measure phagocytosis of neutrophils by flow cytometry, wound cells were stained with Ly6C-FITC and F4/80-APC to identify macrophages and with Ly6G-V450 (clone 1A8; BD Bioscience) to exclude neutrophils. Cells were fixed and permeabilized as described above, then intracellularly stained with Ly6G-PE (clone 1A8; BD Biosciences). Doublets were excluded from analysis and isotype controls for surface and intracellular Ly6G antibodies were used to ensure specificity of intracellular Ly6G staining.

Anti-mouse Ly6G (300 μg; clone IA8; BP0075-1; BioXCell) or its isotype control rat IgG2a (clone 2A3; BP0089; BioXCell) in 1× PBS was administered via the i.p. route to LPS/saline mice. Antibody injections began the same day as LPS/saline injections and continued every 48 h. CFU numbers were assessed at 7 days of infection. Single-cell suspensions were isolated at day 0 (uninfected) and analyzed by flow cytometry to assess depletion efficiency. Intracellular Ly6G was used in place of surface Ly6G to account for potential surface antigen masking by the depletion antibody (70 (link)). After 2% PFA fixation, intracellular Ly6G was stained in the intracellular staining permeabilization wash buffer (BioLegend) using the manufacturer’s protocol. Ly6G antibodies (Ly6G, PE; clone 1A8; BD Pharmingen) used for intracellular staining were diluted half of what was typically used for surface staining.

Leukocyte recruitment into lung tissue was analyzed by flow cytometry, and 100 μl containing 2 × 10⁵ cells from homogenized lung tissue were used for each stain. Cells were centrifuged at 400 g for 2 min and resuspended in staining medium containing PBS, 4% bovine serum albumin (Sigma), and 0.1% sodium azide (Sigma). The following antibodies specific to mouse surface markers were used: Ly6G-PE (clone 1A8, BD Biosciences), CD11b-APC (BD Biosciences), and CD11c-PerCP (BD Biosciences). Isotype controls (BD Biosciences) and Fluorescent Minus One (FMO) control stains were performed. Flow cytometry analysis was performed on a FACSVerse (BD Biosciences) and analyzed with FlowJo software (Tree Star). The total leukocyte population was gated based on FSc and SSc and neutrophils were analyzed based on their specific expression of Ly6G and CD11b (Ly6G^highCD11b^hiCD11c^lo population). Differential cell counts were calculated based on the percentage of neutrophils within the total leukocyte population.

Anti-mouse Ly6G (300 μg; clone IA8; BP0075-1; BioXCell) or its isotype control rat IgG2a (clone 2A3; BP0089; BioXCell) in 1× PBS was administered via the i.p. route to LPS/saline mice. Antibody injections began the same day as LPS/saline injections and continued every 48 h. CFU numbers were assessed at 7 days of infection. Single-cell suspensions were isolated at day 0 (uninfected) and analyzed by flow cytometry to assess depletion efficiency. Intracellular Ly6G was used in place of surface Ly6G to account for potential surface antigen masking by the depletion antibody (70 (link)). After 2% PFA fixation, intracellular Ly6G was stained in the intracellular staining permeabilization wash buffer (BioLegend) using the manufacturer’s protocol. Ly6G antibodies (Ly6G, PE; clone 1A8; BD Pharmingen) used for intracellular staining were diluted half of what was typically used for surface staining.

Macrophages were enriched from isolated wound cells by negative selection. Specifically, cells were stained with Ly6G-PE (clone 1A8; BD Biosciences), CD2-PE (clone RM2–5; BD Biosciences), TER119/Erythroid Cells-PE (clone Ter-119; BD Biosciences), and/or Siglec-F (MerTK^−/− experiments; clone E50–2440; BD Biosciences) for ≤1 hour, washed, and incubated with anti-PE magnetic beads (Miltenyi Biotec) for 20 minutes. Cells were washed and eluted through a magnetic column (Miltenyi Biotec) to deplete non-monocyte/macrophage populations. Enriched monocytes/macrophages were assessed for viability by trypan blue exclusion and surface stained with Ly6C-FITC and F4/80-APC as described above. Eosinophils were identified as F4/80^intSSC^hi and were excluded during sorting. Ly6C^hiF4/80⁺ and Ly6C^lowF4/80^hi populations were sorted to greater than 90% purity under sterile conditions on a FACSAria (BD Biosciences). Sorted cells were assessed for viability by trypan blue exclusion, resuspended in complete medium (DMEM/5% FCS/Penicillin-Streptomycin) and cultured at 37°C/5% CO₂.

For haematoxylin and eosin (H&E) staining, whole ears were fixed in 10% formalin (Sigma) for 24 h and dehydrated into xylene (Pronalys) overnight in a tissue processor (Thermo Fisher Citadel 200). Paraffin wax embedded tissue was sectioned on a microtome at 3–4 μm onto adhesive microscope slides (Trajan). Sections were rehydrated and stained with hematoxylin and eosin as per manufacturer's instructions (Thermo Fisher). Sections were observed using an Olympus BX51TF compound microscope using a 10x, N.A. 0.3 objective, and images taken in the middle of the ear section.
For confocal microscopy, samples were processed and stained using a standard immunofluorescence protocol (37 (link)). Whole ears were incubated in 20% sucrose for 1 h and snap-frozen in OCT compound (Tissue-Tek) using a Stand-Alone Gentle Jane snap-freezing system (Leica Biosystems). Cryosections of 10 μm were fixed in cold acetone for 3 min and blocked with Fc Block (clone 2.4G2) for 1 h and stained with Ly6G-PE (clone 1A8, Pharmingen). For nuclear staining, sections were incubated with DAPI (2 mg/ml) for 10 min. Images were taken with an inverted IX 83 inverted microscope equipped with a FV1200 confocal head (Olympus) using a 20X, N.A. 0.75 objective. Images were acquired using the FV10-ASW software (v4.2b, Olympus) and processed with ImageJ (38 (link)).

Macrophages from the day 14 wound were enriched through negative selection on a magnetic column (Miltenyi Biotec) using the following depletion cocktail: Ly6G-PE (clone 1A8, BD Biosciences), CD2-PE (clone RM2–5; BD Biosciences), Siglec-F-PE (clone E50–2440; BD Biosciences) and TER119/Erythroid Cells-PE (clone Ter-119; BD Biosciences). Staining and column enrichment were carried out as previously described. Following enrichment, wound macrophages were surface stained with Ly6C-FITC, Ly6G-PE, Siglec-F-PE and F4/80-APC for 30 minutes on ice. Sytox Blue (Invitrogen) was used for dead cell exclusion. Ly6C^hiF4/80⁺Ly6G^–Siglec-F^– and Ly6C^low/intF4/80⁺Ly6G^–Siglec-F^– subsets were sorted to ≥90% purity under sterile conditions using a FACSAria.
RNA was isolated from sorted cells using the RNeasy Micro Kit (Qiagen) and cDNA was synthesized with the High Capacity RNA-to-cDNA Kit (Life Technologies). Gene expression analysis was performed by real-time quantitative PCR on a ViiA 7 system (Life Technologies) using TaqMan Assays (Life Technologies) and TaqMan Gene Expression Master Mix (Life Technologies). TaqMan Assay IDs for individual genes are shown in Table S1. Hprt and 18 s served as endogenous controls. Both reference genes produced similar results after normalization. Results using Hprt are shown here. Analysis was carried out using the 2^–ΔΔCt method.