Isolation of CD34+ Cells from Mononuclear Cells (MNCs) from Human Bone Marrow, Peripheral Blood or Cord Blood

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:

• Magnet: See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations
• Mixer allowing both tilting and rotation
• Buffer: PBS (without Ca²⁺ and Mg²⁺) w/0.1% BSA and anticoagulant, pH 7.4
• DNase I (when required)
• Culture Medium: e.g. RPMI 1640 with 2% Fetal Calf Serum (FCS)
• Density gradient medium for MNC (1.077g/l).

Note:

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

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).

• 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.

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.

Detachment Procedure

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


9. 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).


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


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


12. 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.


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


14. Resuspend the cells in Buffer or Culture Medium.


15. 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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