Cd123 pe clone 9f5

The cytotoxic response to DDA of AML cells was determined by flow cytometry. Cells were exposed to DDA for 24 or 48 h then washed with cold phosphate-buffered saline (PBS) and resuspended in 200 µl of Annexin-V binding buffer. For the analysis of AML patient samples, cells were incubated with antibodies against the surface markers CD45-V450 (clone H130), CD34-PeCy7 (clone 8G12), CD38-APC (clone HB7) and CD123-PE (clone 9F5) (BD Pharmagen) before being resuspended in Annexin-V binding buffer. Annexin-V-fluorescein isothiocyanate (BD Pharmagen, clone 2331) and 7-aminoactinomycin D (7AAD) were then added, and the samples were incubated in the dark at room temperature for 15 min. The percentage of viable cells, Annexin-V-7AAD-negative cells, was scored using a flow cytometer.

Trained and control cells were harvested, blocked using block buffer (PBS, 5% rabbit serum, 0.5% goat serum albumin, 5 mM EDTA, 0.1% NaN₃) and stained for flow cytometry using the following mouse anti–human antibodies: CD14 FITC, Clone TÜK4 (Bio–Rad MCA1568F), HLA–DR PerCP–Cy5.5, Clone G56–6 (BD Biosciences 560652), CD123 PE, Clone 9F5 (BD Biosciences 555644), CD11b/Mac–1 PE–Cy7, Clone ICRF44 (BD Biosciences 557743), CD40 APC, Clone 5C3 (BD Biosciences 555591), CD16 Pacific Blue™, Clone 3G8 (BD Biosciences 558122). All flow cytometry experiments were performed on a BD LSRFortessa flow cytometer. Cells were gated by forward scatter, side scatter, and CD14 positivity (Figure S1B). Cell gating was adjusted to account for glucan particles that remained from the initial treatment, given that some residual particles remain after multiple wash steps (Figure S1A). Mean fluorescence of isotype controls was subtracted from sample mean fluorescence to normalize between experimental replicates.