Cd163 pe clone ghi 61

2 x 10⁶ cells were resuspended in 1 mg/mL beriglobin in PBA-E, incubated for 5 min and subsequently stained according to manufacturers recommendation with following antibodies in PBA-E in darkness at 4°C for 20 min. Set 1: CD45 V500 (clone HI30, BD Biosciences), CD15 BV605 (clone W6D3, BD Biosciences), CD14 FITC (clone M5E2, BD Biosciences), CD163 PE (clone GHI/61, BD Biosciences), CD32b PE/Cy7 (clone FUN-2, Biolegend), CD16 PerCP-Cy5.5 (clone 3G8, BD Biosciences), CD206 Alexa Fluor 700 (clone 15-2, Biolegend) and CD64 APC-H7 (clone 10.1, BD Biosciences); Set 2: CD45 V500 (clone HI30, BD Biosciences), FceRI BV605 (clone AER-37 (CRA1), BD Biosciences), CD141 FITC (clone JAA17, ThermoFisher (eBioscience)), CD303 PE/Cy7 (clone 201A, Biolegend), CD1c PerCP-Cy5.5 (clone F10/21A3, BD Biosciences), CD11c APC (clone N418, Biolegend), HLA-DR Alexa Fluor 700 (clone G46-6 (L243), BD Biosciences), CD117 APC/Cy7 (clone 104D2, Biolegend). Cells were washed with PBA-E and analysed on a FACS Canto-Plus flow cytometer (Becton Dickinson). Prior analysis, 1 µg/mL DAPI (4′,6-diamidino-2-phenylindole, Cell Signaling Technology) was added to each sample. Data were analysed with FlowJo V10.7 (BD).

Macrophages and cancer cells were incubated with accutase (eBioscience, Affymetrix, Santa Clara, CA, USA) at 37 °C for 30 min and harvested by gently scraping. Cells were washed and resuspended in FACS buffer (PBS, 2% FBS (Biowest), 0.01% sodium azide) containing appropriate conjugated antibodies, and stained in the dark for 40 min at 4 °C. Macrophages were immunostained with the following antibodies: anti-human CD14-APC (clone MEM-12), CD14-PE/FITC (clone 18D11), CD14-PerCP/Cy5.5 (clone OFC14D), CD86-FITC (clone BU63), HLA-ABC-PE (clone W6/32), HLA-DR-PE/FITC (clone MEM-12) (Immunotools), CCR7-PerCP/Cy5.5 (G043H7), SIRPα-APC (clone SE5A5) (Biolegend, San Diego, CA, USA), CD163-PE (clone GHI/61) (BD Bioscience, Franklin Lakes, NJ, USA). Cancer cells were stained with anti-human CD47-FITC (clone MEM-122) (Immunotools). Unstained cells and single stained with antibodies or with the respective isotype IgGs were used as control. After additional washing steps, cell fluorescence was acquired on a FACS Canto Flow Cytometer (BD Biosciences) using FACS Diva Software. Data analysis was performed with FlowJo software.