Lineage fitc

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).

A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.

Mobilised peripheral blood samples were thawed, washed in PBS and incubated at 37°C with PBS/1% FBS/1 mM EDTA (FACS Buffer) for 30 mins and non-adherent cells collected to remove monocytes. Lineage-negative cells were collected by using EasySep® Human Progenitor Cell Enrichment Kit (STEMCELL Technologies, Victoria, Australia). Cells were incubated with EasySep® Enrichment Cocktail followed by EasySep® Magnetic Nanoparticles. Cells were then incubated for 10 mins in the EasySep® magnet, where lineage-negative cells were magnetically separated. Lineage-negative cells were stained with the following fluorophore-conjugated antibodies: Lineage-FITC, CD34-phycoerythrin (PE)Cy7, CD38-PerCP-Cy5.5, CD45RA-PacificBlue, CD90-APC, CD123-PE (BD Biosciences, San Jose, CA, USA). Cells were FACS sorted and analyzed using BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA). Cells were collected in sterile FACS buffer, and stored at -80°C in QIAzol (Qiagen, Valencia, CA, USA) until RNA extraction.