Hcd45 pecy7

For analysis and sorting of AML and HSPCs derived from hCB or adult BM, cells were stained with hCD45-PeCy7(Clone: H30, cat: 560915, dilution 1 in 25), mCD45-PerCPCy5.5 (Clone:30-F11, cat: 550994, dilution 1 in 400), Lineage-FITC (lin1, cat: 340546, 1 in 25), CD34-PE (Clone 581, cat: 560941, dilution: 1 in 25), and CD38-APC (Clone HIT2, cat: 555462; dilution: 1 in 25) (BD Biosciences, Oxford, UK). Human grafts in mice were assessed using CD19-FITC (Clone: H1B19; cat: 555412, dilution: 1 in 25), CD33-PE (Cat: WM53, cat: 555450, dilution 1 in 25), CD3-APC (Clone: UCHT1, cat: 561811, dilution: 1 in 25), hCD45-PeCy7 (Clone: H30, cat: 560915, dilution 1 in 25), and mCD45-PerCPCy5.5 (Clone:30-F11, cat: 550994, dilution 1 in 400) (BD Biosciences). Luciferase-transduced HL60 and U937 cells were identified and sorted based on their GFP expression. Non-viable cells were excluded by DAPI staining. Appropriate isotype-matched antibodies were used as controls. Flow cytometry analysis was performed using an LSRII flow cytometer (BD Biosciences). Cell sorting was performed using a FACS Aria or INFLUX (BD Biosciences).

Peripheral blood, bone marrow, and spleen were collected from moribund mice. Peripheral blood was stained with hCD3 FITC (BD 349201), hCD19 PE (BD 340720), hCD45 PeCy7 (BD 557748), and mCD45 APC (BD 553991) and analyzed using FACS LSR II BD (BD Biosciences). Upon sacrifice, xenografted cells from the bone marrow and spleen were separated from murine cells by staining with hCD45 microbeads followed by negative selection of murine cells with LS columns according to manufacturer’s protocol (MACS Miltenyi Biotec), and frozen for subsequent drug sensitivity studies. For a given patient sample, cells from each mouse were pooled for screening.