Human cd34 cd38 isolation kit

Bone marrow samples were collected from patients newly diagnosed with CML as part of an institutionally approved protocol for cell sample collection (Centre Hospitalier Nice, 06204 Nice, France). Cord blood and bone marrow samples were collected from healthy donors with informed consent. Mononuclear cells were isolated by density centrifugation (Ficoll-Paque Plus, STEMCELL Technologies), washed with PBS with 5% fetal calf serum (FCS). For clonogenic assays CD34⁺ cells were positively enriched with CD34 microbeads (CD34 MicroBead Kit, Miltenyi Biotec). For LTC-IC, CD34⁺ cells were first positively enriched and then depleted for CD38⁺ cells (human CD34⁺CD38⁻ isolation kit, Miltenyi Biotec). The CD34⁺ cells, after sorting, were stained with anti-human-CD34 antibody (Miltenyi Biotec, 130-113-741) and checked by flow cytometry to ensure purity (always above 96%).

UCB and granulocyte CSF (G-CSF) MPB were obtained using CCHMC Institutional Review Board–approved protocols, and CD34⁺ HSPC were isolated by magnetic selection with the human Indirect CD34 Microbead kit (Miltenyi Biotec, 130-046-701). CD34⁺ purity was > 95% as confirmed by flow cytometry. CD34⁺CD38^– cells were isolated from G-CSF MPB by magnetic selection with human CD34⁺CD38^– Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved. Thawed CD34⁺ or CD34⁺CD38^– cells were cultured in X-VIVO 10 (Lonza, BE02-055Q) or SCGM (CellGenix, 20806-0500) supplemented with 2% human serum albumin, 100 ng/mL TPO, 300 ng/mL SCF, and 300 ng/mL FLT3-L (all cytokines purchased from Peprotech) at a cell density of 2 × 10⁶ to 5 × 10⁶ CD34⁺ cells/mL. Media was supplemented with Birb 796 (600nM) (Selleckchem) throughout culture and PGE2 (10 μM) (Cayman) at plating, transduction, and 1 hour before harvest. Cells were transduced with a LV vector in culture for 36–42 hours where indicated. CD34⁺ HSPC were transduced at a final concentration of 5 × 10⁷ to 1 × 10⁸ IU/mL. Following culture, CD34⁺ HSPC were harvested and washed with PBS, resuspended in PBS, and used in in vivo or in vitro experiments, as described below.