F4 80 pe cy7

The retinal cells were stained with fluorescent dyes conjugated to antibodies as follows: CD206-FITC (C068C2; BioLegend, San Diego, CA, USA); 7-AAD (BD Pharmingen, San Jose, CA, USA); CD11b-APC-Cy7 (M1/70; BD Biosciences San Jose, CA, USA); f4/80-PE-Cy7 (BM8; BioLegend). After staining, the cells were washed twice with a FACS buffer (0.5% BSA in PBS buffer) and suspended in the FACS buffer for FACS analyses. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software, Los Angeles, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages.
To investigate intracellular cytokine production, retinal cells were treated with a leukocyte activation cocktail with BD GolgiPlug^TM (BD Biosciences) for 4.5 h and with a BD Cytofix/Cytoperm Fixation/Permeabilization^TM kit (BD Biosciences) and then stained with the following antibodies: TNF-α-FITC (MP6-XT22; eBiosciences, San Diego, CA, USA); IL-10-PE (GK1.5; BioLegend); CD11b-APC-Cy7 (M1/70; BD Biosciences); f4/80-PE-Cy7 (BM8; BioLegend); and 7-AAD (BD Pharmingen, San Jose, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software).

Leukocyte infiltrates into the surrounding soft tissue following S. aureus PJI were characterized using flow cytometry as previously described (59 (link)). Briefly, the soft tissue surrounding the knee joint was excised, disrupted using the blunt end of a 3-ml syringe plunger in PBS containing protease inhibitor (Thermo Scientific, Rockford, IL), and passed through a 70-μm filter. Red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend, San Diego, CA), and the single-cell suspension was stained with CD11b-fluorescein isothiocyanate (FITC), CD45-allophycocyanin (APC), Ly6G-phycoerythrin (PE), Ly6C-peridinin chlorophyll protein (PerCP)-Cy5.5, F4/80-PE-Cy7 (BioLegend and BD Biosciences, San Diego, CA), and a Live/Dead fixable blue dead cell stain kit (Invitrogen, Eugene, OR) according to the manufacturers’ instructions. Cell populations were analyzed using a BD LSR II and FACSDiva software (BD Bioscience, San Jose, CA), where MDSCs (CD11b^high Ly6G⁺ Ly6C⁺ F4/80⁻), neutrophils (CD11b^low Ly6G⁺ Ly6C⁺ F4/80⁻), monocytes (Ly6G⁻ Ly6C⁺ F4/80⁻), and MΦs (Ly6G⁻ Ly6C⁻ F4/80⁺) are reported as the percentage of live CD45⁺ cells.

Single-cell suspensions from the SVF of fat tissue, spleen, and blood samples were incubated in FcBlock (BD Biosciences) for 15 min at 4°C. The cells were then stained with fluorophore-conjugated antibodies for 20 min at 4°C in the dark. The antibodies were purchased from BD Biosciences (CD8-FITC, CD11c-FITC, NK1.1-FITC, CD4-PE/Cy7, CD45.2-APC), eBioscience (San Diego, CA) (NKG2D-PE, F4/80-PE/Cy7, CD11b-APC/eFluor780), or BioLegend (San Diego, CA) (CD3ε-Pacific Blue). Dead cells were excluded with 7-AAD staining (BD Biosciences). Flow cytometry data was acquired with FACSCantoII or LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo (Treestar, Ashland, OR).