Cd45 v500 clone hi30

The following stains/antibodies were used for surface staining: BD Horizon™ Fixable Viability Stain 450 (BD Biosciences, San Jose, CA, USA), CD45-V500 (clone HI30, BD Biosciences), CD14- APC-Cy7 (clone M5E2, BioLegend, San Diego, CA, USA), CD117-APC (clone 104D2, BD Biosciences), FcεRIα-PE and FcεRIα-FITC (clone AER-37 (CRA-1), BioLegend), CD34-Pe-Cy7 (clone 581, BD Biosciences), Integrin-β7-FITC (clone FIB504, eBioscience), MrgX2-PE (clone K125H4, BioLegend), and CD63-Pe-Cy7 (clone H5C6, BD Biosciences). To measure proliferation, the cells were labeled with CellTrace™ Far Red (Thermo Fisher Scientific) prior to treatment. Pure human lung mast cells were obtained by FACS of CD45⁺CD14⁻CD117^high cells using a FACSAria I instrument, flow cytometric analyses were performed with a FACSCanto II instrument (BD, Franklin Lakes, NJ, USA), and flow cytometry data analysis was performed with FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA).

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).