Anti cd117 apc

Whole blood stainings were performed within 24 h after blood collection. Leukocytes were counted using Cell-Dyn Sapphire Hematology System (Abbott Diagnostics, Chicago, IL, USA). For staining, 1 million cells per tube were used based on direct blood cell count. Directly labeled antibodies were added to whole blood and incubated for 20 min at 4 °C, followed by 10 min red-blood-cells lysis (Bühlmann Laboratories, Schönenbuch, Switzerland) and subsequently washed using cold PBS. All anti-human antibodies were used at the concentrations recommended by the manufacturer: anti-CD15-PeCy7 (clone HI98), anti-CD14-Pe (clone MφP9), anti-CD163-FITC (clone GHI/61), anti-CD11b-BV510 (clone ICRF44), anti-CD33-V450 (clone WM53), anti-CD64-APCH7 (clone 10.1), anti-CD117-APC (clone YB5.B8), anti-CD45RA-PeCy7 (clone HI100), anti-CD25-Pe (clone M-A251), anti-CD4-FITC (clone RPA-T4), anti-CD8-V500 (clone SK1), anti-CD45RO-BV421 (clone UCHL1), anti-CD3-APCH7 (clone SK7), and CD127-Alexa Fluor 647 (clone HIL-7R-M21) and 7AAD (all from Becton Dickinson). BD FACSCanto II (Becton Dickinson) instrument was used to analyze samples and FlowJo 10.6.2 (Treestar Inc., Ashland, OR, USA) software and several software plugins (FlowCLEAN, downsample_V3, FlowSOM, tSNE) were used to analyze all data.

The BALF were incubated with different antibodies for 1 h: FITC anti-CCR3, PE anti-Siglec-F for eosinophil [43 ], FITC anti-FcεRIα, and APC anti-CD117 for mast cells, FITC anti-CD11c, and APC anti-CD40 and APC anti-CD86 for DCs (BD Biosciences, New Jersey, USA or Biolegend, California, USA). After the BALF were washed by PBS three times, the different types of cells were analyzed and sorted using a flow cytometer (BD FACSAria, USA) and data were analyzed using CellQuest software (BD Biosciences, USA).