Introduction

This product is intended for positive magnetic isolation of CD34+ cells from mononuclear cells (MNCs) from human bone marrow, peripheral blood or cord blood. In the first step CD34+ cells are captured by the Dynabeads® and isolated using a Dynal® magnet. In the second step beads are removed from the cells

Downstream applications

Isolated cells are bead- and antibody-free, phenotypically unaltered and suitable for any downstream application including flow cytometry, functional studies and cell culture. For recommended products and protocols visit Immunology Research Guide.

Description of materials

Materials Supplied

Dynabeads® CD34 are uniform, superparamagnetic polymer beads (4.5 μm diameter) coated with a primary monoclonal antibody specific for the CD34 membrane antigen predominantly expressed on human hematopoietic progenitor cells and endothelial progenitor cells. The Dynabeads® CD34 are supplied in PBS w/ 0.1% BSA and 0.02% NaN3. DETACHaBEAD CD34 is a polyclonal anti-Fab antibody specific for the CD34 antibody on the Dynabeads® CD34.

Additional Materials Required

Materials that are not included but are needed to perform the entire protocol:


Note
:

  • BSA can be replaced by human serum albumin (HSA) or FCS.
  • Anticoagulant: 0,6% citrate or 2 mM EDTA.

Ordering Information

Catalog # Name Size List Price (USD) Qty
11301D Dynabeads® CD34 Positive Isolation Kit 5 mL 1,026.00

Protocol

This protocol describes isolation of CD34+ cells from 1 ml MNC at starting concentration 4 x 107 - 1 x 108 MNCs/ml. Use 4 x 107 (100 μl) Dynabeads® CD34; for scale-up to larger cell numbers, adjust the volumes accordingly.

Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

  1. Resuspend the Dynabeads® in the vial.

  2. Transfer the desired volume of Dynabeads® to a tube.

  3. Add the same volume of Buffer, or at least 1 ml, and mix.

  4. Place the tube in a magnet for 1 min and discard the supernatant.

  5. Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer as the initial volume of Dynabeads® (step 2).

Sample Preparation

Prepare an MNC suspension following normal procedures. Always use fresh samples. When working with cord blood or bone marrow samples, DNase treatment is recommended: Please visit www.invitrogen.com/cellisolation and follow our QuickLinks for Sample Preparation details.

Critical Steps for Cell Isolation

  • Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads® do not settle at the bottom of the tube.
  • When incubating Dynabeads® and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.
  • PBS containing Ca2+ or Mg2+ is not recommended, except when required for DNase treatment.

Isolation Procedure

  1. Resuspend the prepared cell sample thoroughly with a narrow pipette.

  2. Add 100 μl Dynabeads® to the prepared sample and vortex 2-3 seconds.

  3. Incubate at 2 - 8°C for 30 min. with gentle tilting and rotation.

  4. Fill the tube with cold Buffer to the height of the magnet (or at least 1 ml) and resuspend the cell-bead complexes.

  5. Place the tube in a magnet for 2 min. and discard the supernatant.

  6. Resuspend the bead-bound cells in 2 ml Buffer by vortexing or pipetting. Separate on a magnet for 1 min.

  7. Repeat step 6 twice.

  8. Resuspend the bead-bound cells in 100 μl Buffer.

  9. Detachment Procedure

    Tubes and Buffer can be kept at room temperature for the detachment procedure.

  10. Add 100 μl DETACHaBEAD. (Never use less than 100 μl DETACHaBEAD.)

    Incubate at room temperature for 45 min. with tilting and rotation. (Note: Keep the sample in the bottom of the tube).

  11. Add 2 ml Buffer and vortex 2-3 seconds to enhance detachment of beads from the cells.

  12. Place the tube in a magnet for 2 min.

  13. Transfer the supernatant containing released cells to a fresh tube. To obtain residual cells, wash the beads 3 times in 500 μl Buffer and pool the supernatants.

  14. Wash the cells thoroughly by resuspending in 10 ml Buffer. Centrifuge for 10 min at 400 x g to remove excess DETACHaBEAD.

  15. Resuspend the cells in Buffer or Culture Medium.

  16. The isolated cells are pure, viable and free from antibody bound to the surface, and may be used in any downstream application.

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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

 PBMC:

  1. Mändle T et al. (2005) Infection of human CD34+ progenitor cells with Bartonella henselae results in intraerythrocytic presence of B henselae. Blood 106(4): 1215-1222.

  2. Ouriaghli FE et al. (2003) Clonal dominance of chronic myelogenous leukemia is associated with diminished sensitivity to the antiproliferative effects of neutrophil elastase. Blood 102(10): 3786-3792.

  3. Spencer A et al. (2001) Enumeration of bone marrow ‘homing’ haemopoietic stem cells from

  4. G-CSF-mobilised normal donors and influence on engraftment following allogeneic transplantation. Bone Marrow Transplantation 28: 1019–1022.

Cord blood:

  1. Tanaka N et al. (2004) Constitutive Expression and Involvement of

  2. Cyclooxygenase-2 in Human Megakaryocytopoiesis. Arterioscler. Thromb. Vasc. Biol. 24: 607-612.

  3. Tsutsuda-Asano A et al. (2000) Transduction of fibroblasts and CD34+ progenitors using a selectable retroviral vector containing cDNAs encoding arylsulfatase A and CD24. J. Hum. Genet. 45:18-23.

  4. Huard B et al. (2004) BAFF production by antigen-presenting cells provides T cell co-stimulation. Internat Immunol. 3:467-475.

  5. Shima Y et al. (2006) L-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes. Blood. 107:1352-1356.

  6. Gudmundsson KO et al. (2007) Gene-expression analysis of hematopoietic progenitor cells identifies Dlg7 as a potential stem-cell gene. Stem Cells. Doi:10.1634/stemcells.2005-0479.

Bone marrow (BM):

  1. Zannettino ACW et al. (2003) Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration. Biochem. J. 370:537-549.

  2. Hirohata S et al. (2002) Bone marrow CD34 progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD B cells through direct cellular interaction. Journal of Leukocyte Biology 71:987-995.

  3. Myklebust JH et al. (2000) Protein kinase C-aisoform is involved in erythropoietin-induced erythroid differentiation of CD34+ progenitor cells from human bone marrow. Blood, 95(2):510-518.

  4. Nakamura T et al. (2006) A novel nonpeptidyl human c-Mp1 activator stimulates human megakaryopoiesis and thrombopoiesis. Blood. 107: 4300-4307.

  5. Døsen G et al. (2006) Wnt expression and canonical Wnt signaling in human bone marrow lymphopoiesis. BMC Immunol. Biomedcentral. com/1471-2172/7/13.

  6. Goselink HM et al. (2006) Cytokine-Dependent Proliferation of Human CD34+ Progenitor Cells in the Absence of Serum is Suppressed by Their Progeny’s Production of Serine Proteases. Stem Cells. 24:299-306.

Endothelial Cell Isolation:

  1. Aasheim H-C et al. (2005) Ephrin-A1 binding to CD4+ T lymphocytes stimulates migration and induces tyrosine phosphorylation of PYK2. Blood. 105:2869-2876.

  2. Pellet C et al. (2006) Kaposi’s Sarcoma-Associated Herpesvirus Viremia is Associated with the Progression of Classic and Endemic Kaposi’s Sarcoma. J Invest Dermat.126:621-627.


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113.01D.indd   Rev 003     5-May-2007