Ly6g phycoerythrin

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.

Heart tissues were analyzed by flow cytometry as previously described (64 (link)). Briefly, hearts were digested with digestion solution containing collagenase II (1.5 mg/ml; A004174, Worthington), collagenase IV (1.5 mg/ml; A004186, Worthington), and deoxyribonuclease I (60 U/ml; A3778, AppliChem, Lochem, Darmstadt, Germany) in Hank’s balanced salt solution. Cardiac single-cell suspensions were gained by filtering digested tissues through 70-μm cell strainers (BD Biosciences, San Jose, CA, USA). Single-cell suspensions were blocked with Fc block and then stained with CD45-PE-CY7 (103114, BioLegend), CD11b–fluorescein isothiocyanate (101205, BioLegend), CD64-allophycocyanin (139305, BioLegend), Ly6G-phycoerythrin (127607, BioLegend), and CD3-PE (100205, BioLegend). Samples were analyzed using LSRFortessa (BD Biosciences).