Anti cd123

PBMCs were incubated with fluorochrome-conjugated mAbs and analyzed on either an FC 500 (Beckman Coulter) or an LSRFortessa (Becton Dickinson) flow cytometer. FITC-, PE-, PE-Texas Red-, ECD-, APC-, PE-Cy5–, PE-Cy7–, PerCP-Cy5.5–, Pacific Blue-, and AF700-conjugated mouse anti-human mAbs included anti-CD3, anti-CD4, anti-CD8, anti-CD11c, anti-CD14, anti-CD16 (clone 3G8), anti-CD19, anti-CD25, ant-CD28, anti-CD45RA, anti-CD45RO, anti-CD80, anti-CD86, anti-CD123, anti-CTLA-4, anti–HLA-DR, anti-IL2, anti-Ki-67 (BD Pharmingen), anti-CD56, anti-CD83 (Beckman Coulter), anti-CD127, anti- IFNγ, anti-LAG-3, anti-PD-1, anti- TNFα (eBioscience), anti-CCR7, anti-TIM-3 (R&D Systems), and anti-CD57 (BioLegend). Nonreactive isotype-matched antibodies (Becton Dickinson, eBioscience, R&D Systems) were used as controls. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) facilitated exclusion of dead cells. Gates were set for collection and analysis of at least 20,000 live events. Data were analyzed with FlowJo 9.5 software (TreeStar).

Fluorochrome-conjugated isotype controls, anti-CD 123, antiCLEC12A, anti-His, and anti-CD3 were purchased from BD Biosciences. Recombinant human CD123-His was produced by Sino Biological. Analysis was performed on at least 20,000 cells per sample using an Attune NxT instrument (Thermo Fisher Scientific) and analyzed with Flojo software. Antigen quantification was performed using QuantiBrite beads (BD Biosciences) per manufacturer protocol. Each quantification was performed in triplicate.