Hla abc pe

First, supernatant was removed and stored for CBA analysis, and then wells were carefully washed with PBS and 10 mM EDTA/PBS was added for 5 min at 37°C. Cells were subsequently gently harvested using a cell lifter. For the FACS staining, macrophages were blocked with the FcR blocking reagent, human (Miltenyi) prior to surface staining for CD80 APC-H7 (L307.4, BD), CD86 FITC (FM95, Miltenyi), HLA-ABC PE (REA230, Miltenyi), HLA-DR APC (AC122, Miltenyi), CD163 PerCPCy5.5 (GHI/61, BD), CD206 APC (19.2, BD), CD209 PerCPCy5.5 (DCN46, BD), and PD-L1 PE-Cy7 (REA1197, Biolegend) for 30 min in the dark and on ice. Directly before measuring the sample on the flow cytometer, 30 ng DAPI (Sigma-Aldrich) was added. To control for background staining, cells stained only with 30 ng DAPI were used. All flow cytometric analyses were performed with BD Canto II and data were analyzed with FlowJo 10.1r5 64-bit software.

To determine HER2 and HLA surface expression, SKBR3 and MCF7 cells were stained with Live/Dead^® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) for 30 min on ice, followed by staining with HER2-APC (Neu24.7, BD), HLA-C-PE (DT9, BD), HLA-Bw4-PEVio770 (REA274, Miltenyi), HLA-E-PE (3D12, Thermo Fisher Scientific), HLA-ABC-APC (G46-2.6, BD), or HLA-ABC-PE (REA230, Miltenyi), or matched isotype controls for 30 min on ice. All analysis by flow cytometry was performed with BD FACS Canto II. Data were analyzed with FlowJo v10.6.1 64-bit software, (TreeStar, Ashland, OR, USA).