Cd15 bv510

Fc receptor blocking solution (BioLegend, USA) was added prior to staining. The following antibodies were used in this study: CD45-APC-H7 (BD Bioscience, USA), CD45-Percp-Cy5.5 (BD Bioscience, USA), CD33-PE-Cy7 (BioLegend, USA), CD11b-FITC (BioLegend, USA), CD11b-BV421 (BD Bioscience, USA), CD11b-APC (BioLegend, USA), HLA-DR-BV421 (BD Bioscience, USA), HLA-DR-FITC (BioLegend, USA), CD14-PE (BD Bioscience, USA), CD15-APC (BD Bioscience, USA), CD15-BV510 (BioLegend, USA), CD3-FTIC (BioLegend, USA), IFN-γ-PE-Cy7 (BioLegend, USA), CD4-APC (BioLegend, USA), CD8-PE (BioLegend, USA), CD16-PE (BD Bioscience, USA), CD56-PE-Cy7 (BD Bioscience, USA), Fas-APC (BD Bioscience, USA), CD261-APC (BioLegend, USA), CD262-PE (BioLegend, USA), CD263-PE (eBioscience, USA), and CD264-PE (R&D Systems, USA). FVD eFluor 780 (eBioscience, USA) was used to identify dead cells for excluding them from the analysis. Flow cytometry data were acquired with LSRFortessa (BD Bioscience, USA) or Beckman Coulter FC500 (Beckman, USA), and were analyzed using FlowJo software (BD Bioscience, USA). Positive subpopulations were identified by comparing stained samples with Fluorescence minus one (FMO) controls.

PBMCs from a subset of patients undergoing THA or TKA were analyzed by flow cytometry (n = 19). Within 60 min after each draw, blood was layered over Ficoll-Paque PLUS, leukocytes were collected from the interface, and remaining red blood cells (RBCs) were lysed using RBC Lysis Buffer (BioLegend; San Diego, CA, USA). After lysis, cells were washed, incubated with Human FcR Binding Inhibitor (eBioscience; San Diego, CA, USA), and stained with anti-human CD8a-AlexaFluor488, CD4-PE, CD66b-PE-Dazzle, CD25-APC, CD45-APC-Cy7, CD127-PerCP-Cy5.5, CD14-PE-Cy7, HLA-DR-BV421, CD15-BV510, CD19-BV605, CD33-BV711, and CD16-BV650 (all from BioLegend; San Diego, CA, USA). Dead cells were excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies; Eugene, OR, USA) and analysis was performed using BD FACS-DIVA software, as previously described with the gating strategy depicted in Supplementary Figure S1 [16 (link)].