Flow Cytometry Analysis of PBMCs

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)].