Cd56 b159

MNC from FLT3-ITD AML BM samples were stained for lineage markers using biotinylated antibodies: CD4 (RPA-T4; Biolegend), CD8a (RPA-T8; Biolegend), CD19 (HIB19; Biolegend), CD41 (MEM-06; Sigma), CD235alpha (HIR2; eBioscience), CD56 (B159; BD Pharmingen). Cells were then stained with the following fluorochrome-conjugated antibodies: CD34-FITC (581; BD Pharmingen), CD90-PE (5e10; BD Pharmingen), CD33-PC5.5 (D3HL60, Beckmann Coulter), CD45RA-APC Cy7 (H1100; BD Pharmingen), Streptavidin-eFluor 450 (eBioscience), CD38-APC (HB7; BD Pharmingen), CD45-APC-Cy7 (2D1; BD Pharmingen). PI was added as live/dead marker. Cell sorting was performed on a FACS Aria II (Becton Dickinson, Heidelberg, Germany). Sorting purity of >98% was routinely obtained.

Isolated PBMC were washed once and then incubated with fluorescently-labeled antibodies for 20 min at 4° C in staining buffer (PBS + 0.5% BSA). Antibodies used in this study included reagents specific for: CD3 (UCHT1) and CD56 (B159) from BD Pharmingen (San Diego, CA); VGF (D-20) and the detection antibody, donkey anti-goat IgG FITC, from Santa Cruz Biotechnology (Dallas, Texas, USA). Afterwards, data were collected on a FACS flow cytometer (Fortessa, BD Biosciences, Mountain View, CA) and analyzed using FACS DIVA software 6.1.3 (BD Biosciences, Mountain View, CA) and using FlowJo software (Treestar Inc., Ashland, OR). The data were analyzed using biexponential transformation function for complete data visualization.

Quantitative flow cytometric analyses and FACS of human live MNCs were performed using a previously described gating strategy and antigen panel^{1 (link),15 (link)} and antibodies against CD2 (RPA-2.10; 1:20), CD3 (SK7; 1:10), CD14 (MφP9; 1:20), CD19 (SJ25C1; 1:10), CD20 (2H7; 1:10), CD34 (581; 1:20), CD56 (B159; 1:40), CD123 (9F5; 1:20), and CD235a (HIR2; 1:40; all from BD Biosciences, Franklin Lakes, NJ); CD4 (S3.5; 1:20), CD11b (ICRF44; 1:20), CD33 (P67.6; 1:20), and CD90 (5E10; 1:10; all from Thermo Fisher Scientific, Waltham, MA); CD7 (6B7; 1:20) and CD38 (HIT2; 1:20; both from BioLegend, San Diego, CA); CD10 (SJ5-1B4; 1:20; Leinco Technologies, St. Louis, MO); and CD45RA (HI100; 1:10; Tonbo Biosciences, San Diego, CA).
FACS-purified samples were acquired with a BD Influx Cell Sorter (BD Biosciences), and the cell populations were analyzed using FlowJo software (version 10.7.1, Ashland, OR). All experiments included single-stained controls and were performed at MD Anderson’s South Campus Flow Cytometry and Cellular Imaging Facility.