Cd33 bv711

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)].

Out of the 104 mBC patients included in the mass cytometry analysis, tumor biopsies were obtained from 63 patients (HR⁺ BC, n = 42; TNBC, n = 21) and were subjected to flow cytometry. Briefly, freshly obtained tumor biopsies were mechanically chopped and then dissociated in DMEM F12 medium containing 10% FBS, collagenase-IV (1 mg/mL), hyaluronidase (1 mg/mL), & DNase-I (50 µg/mL) in a 37 °C water bath for 45 min. Single cell suspensions were then filtered, washed with PBS, and stained with antibodies CD45-A488, CD3-Pacific blue, CD4-PE, CD25-BV605, CD14-PE-Cy7, CD33-BV711, HLA-DR-BV650 (Biolegend), CD127-BV786 (BD Biosciences), and Fixable Viability Dye (eFluor780, ThermoFisher). Samples were acquired using LSR-II/FACSymphony A5 flow cytometer (BD Biosciences), and the data analyzed with FlowJo.