Hcd45 microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

HCD45 microbeads are magnetic particles coated with antibodies specific for the CD45 surface antigen. They are designed for the isolation and enrichment of CD45-positive cells from various sample types.

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Lab products found in correlation

Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Recombinant il 3, by R&D Systems (1 mentions) Recombinant il 7, by R&D Systems (1 mentions) Sfem 2 medium, by STEMCELL (1 mentions) Lysing buffer, by BD (1 mentions) Cd38 hit2, by BD (1 mentions)

3 protocols using hcd45 microbeads

Culturing Primary Human ALL Cells with MSCs

Cited 1 time

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Primary human ALL cells were obtained after informed consent at West Virginia University Cancer Center; Cincinnati Children’s Hospital Medical Center Respiration Core; and Pittsburgh Biospecimen Core under the approved Institutional Review Broad (IRB) protocols: #1310105737; STUDY19030357 and # 2011-3023. Primary human ALL cells were cultured on hTERT-immortalized primary BM mesenchymal stromal cells (MSCs)^{39 ,40 (link)}. MSCs were seeded at a density of 10⁴ cells/cm² in MSC medium 48 h prior to adding to ALL. ALL cells were seeded onto MSC at a density of 2 × 10⁶ cells/ml in SFEM II medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 20% fetal calf serum (GIBCO, Life Technologies), 20 ng/ml recombinant IL-3 (R&D Systems, Abingdon, UK) and 10 ng/ml recombinant IL-7 (R&D Systems, Minneapolis, MN). ALL cells were harvested every 7 days. Non-adherent cells present in supernatant medium were washed with PBS and passed through a 15 μm filter (pluriSelect Life Science, Leipzig, Germany). ALL cells were separated from MSCs by magnetic cell separation using hCD45 microbeads (Miltenyi Biotec, Auburn CA). Viable ALL cells were counted by Trypan blue exclusion, re-suspended in fresh ALL medium, and seeded onto fresh MSC.

Wu L., Chatla S., Lin Q., Chowdhury F.A., Geldenhuys W, & Du W. (2021). Quinacrine-CASIN combination overcomes chemoresistance in human acute lymphoid leukemia. Nature Communications, 12, 6936.

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Quantifying Human Hematopoietic Engraftment

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Retro-orbital bleeding was performed at 8 weeks post-transplant to determine the levels of human engraftment. Red blood cells were lysed using the lysing buffer (BD Biosciences, San Jose, CA). The levels of human engraftment were determined by flow cytometry (detailed flow panels in supplementary methods), and calculated as (hCD45⁺/(hCD45⁺+mCD45⁺))×100. Mice were euthanized 16 weeks post-transplant. Two femurs were collected and flushed with MACS buffer. Spleens were squeezed though a 70um filter. Red blood cells were lysed using the lysing buffer (BD Biosciences, San Jose, CA). The levels of human engraftment were determined by flow cytometry. The human cells from mouse BM were isolated using hCD45+ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), gDNA was extracted, and the levels of gene editing in human cells were determined by HTS, as described earlier. Mouse harvest, flow cytometry and sequencing analyses were performed in a blinded manner.

Lomova A., Clark D.N., Campo-Fernandez B., Flores-Bjurström C., Kaufman M.L., Fitz-Gibbon S., Wang X., Miyahira E.Y., Brown D., DeWitt M.A., Corn J.E., Hollis R.P., Romero Z, & Kohn D.B. (2018). Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair. Stem cells (Dayton, Ohio), 37(2), 284-294.

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Engraftment and Multilineage Reconstitution in NSG Mice

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Eight-week-old NSG mice were sublethally irradiated (300 rad) and transplanted with 1 × 10⁶ unedited and edited CD34+ cells. Donor chimerism was assessed every 4 weeks as the frequency of human cells in total white blood cells by flow cytometry after red blood cell lysis and staining with human CD45 (BD Biosciences). Upon sacrifice, 16 or 20 weeks after transplantation, the chimeric bone marrow was harvested and assayed for level of engraftment and multilineage reconstitution after staining with the following antibodies: CD45 (2D1), CD19 (HIB19), CD33 (P67.6), CD34 (8G12), and CD38 (HIT2) (BD Biosciences) and CD41 (VIPL3) (Invitrogen). For the secondary transplantations, bone marrow cells were enriched for human CD45+ cells by immunomagnetic separation (hCD45 microbeads, Miltenyi Biotec), and the same number of human cells (5 × 10⁶) was injected into each secondary recipient. Secondary recipients were sacrificed 10 weeks after transplantation. All in vivo experiments were conducted with approval from the institutional animal care and use committee.

Psatha N., Reik A., Phelps S., Zhou Y., Dalas D., Yannaki E., Levasseur D.N., Urnov F.D., Holmes M.C, & Papayannopoulou T. (2018). Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major. Molecular Therapy. Methods & Clinical Development, 10, 313-326.

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