Direct cd34 microbead kit

After mononuclear cells were separated from human umbilical cord blood, CD34⁺ HSPCs were isolated using a direct CD34⁺ MicroBead kit (Miltenyi Biotec). Three- to four-week-old NSG mice were irradiated with 200 cGy using a ¹³⁷Cs gamma irradiator. Over 90% pure freshly isolated CD34⁺ HSPCs were injected intravenously, 24 h after irradiation, at a density of 1–2 × 10⁵ CD34⁺ cells/mouse. The engraftment levels of human CD45⁺ cells were determined in the peripheral blood, as early as 4 weeks post CD34 injection, by flow cytometric quantification, as well as other human immune populations. Mice with 25% human CD45⁺ cells were considered as humanized (Hu-NSG mice). In-depth analysis of bone marrow and spleen tissue for human immune cell subpopulations, including CD45⁺, CD3⁺, CD4⁺, and CD8⁺ T cells, B cells, NK cells, and lineage-negative cells, was performed using a 10-color flow cytometry panel at weeks 6 and 9 post CD34⁺ engraftment. Hu-NSG mice from different cord blood donors with different levels of engraftment were randomized into every treatment group in all of the experiments. All Hu-NSG mice were verified for humanization before tumor implantation.

HIS-NSG mice (male and female) were generated using the human stem cell neonate NSG protocol as previously described (18 (link), 19 (link)). In brief, human fetal liver was obtained from Advanced Bioscience Resources, California, USA. The tissue was mechanically cut into small pieces with surgical scissors and treated with 2 mg/mL collagenase D (Roche) in Hank's balanced salt solution with CaCl₂/MgCl₂ (Gibco) for 30 min in a 5% CO₂ at 37°C followed by filtering through 70-μm nylon cell strainers (BD Biosciences). CD34⁺ human hematopoietic stem and progenitor cells (HSCs) were isolated using the direct CD34 MicroBead kit (Miltenyi Biotec). One- to three day-old NSG mice were irradiated with 100 cGy and injected intrahepatically with 1–2 × 10⁵ CD34⁺ HPCs 24 h after irradiation. Some NSG mice received 50 μl of PBS as a control. The mice were bled 10–12 weeks after engraftment and peripheral lymphocytes were stained with monoclonal antibodies purchased from BioLegend: anti-mouse CD45 (clone 30-F11), anti-human CD45 (clone HI30), anti-human CD3 (clone UCHT1), anti-human CD4 (clone OKT4), anti-human CD8 (clone SK-1), and anti-human CD19 (clone HIB19) for 30 min at 4°C. After red blood cell lysis, the samples were analyzed by flow cytometry using a BD LSR II to assess reconstitution of the human immune system and visualized using FlowJo software.