Isolate or Deplete CD15+ Cells - Whole Blood, Buffy Coat, MNC Suspensions

Isolate or deplete CD15⁺ cells directly from whole blood, buffy coat or MNC suspensions with Dynabeads CD15. For rapid and consistent results in protein or gene expression analysis, lyse the granulocytes while they are still attached to the beads and directly process for further molecular analysis.

Principle of Isolation

Dynabeads are mixed with the cell sample in a tube. The Dynabeads will bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet.

• Positive Isolation - discard the supernatant and use the bead-bound cells for downstream applications. (e.g. molecular analysis).
  
• Depletion - discard the bead-bound cells and use the remaining, untouched cells for any application.

Description of Materials

Dynabeads CD15 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal mouse IgM antibody recognizing the CD15 membrane antigen. The CD15 antigen is predominantly expressed on human neutrophil and eosinophil granulocytes, and to a varying degree on monocytes (1). The CD15 antigen shows heterogeneous expression on normal myeloid precursor cells, myeloid leukemias, myeloid cell lines and Sternberg- Reed cells. The CD15 antigen is widely distributed outside the hematopoietic system.

5 ml Dynabeads CD15

• 4 x 10⁸ beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN₃).
• This product will process up to 2 x 10⁹ cells

Additional Materials Required

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

Magnet (Dynal MPC™): See www.lifetechnologies.com/magnets-selection for magnet recommendations.

• Mixer allowing both tilting and rotation.
• Buffer 1: PBS w/0.1% BSA and 2 mM EDTA, pH 7.4.

Important Notes:

• BSA can be replaced by human serum albumin (HSA) or Fetal Calf Serum (FCS).
• EDTA can be replaced by sodium citrate.
• PBS containing Ca²⁺ or Mg²⁺ is not recommended.

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 1, 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 1 as the initial volume of Dynabeads (step 2).

Sample Preparation - Whole Blood and Buffy Coat

Blood and buffy coat can be diluted before use to decrease the granulocyte concentration. This is to ensure that
the number of Dynabeads per target cell is higher than 4:1 in granulocyterich samples.

  1.    Dilute whole blood (1+1) in Buffer 1

  2.    Dilute buffy coat (1+4) in Buffer 1

Dynabeads can be added directly to undiluted blood or buffy coat if reduced cell isolation efficiency is tolerated (e.g. rapid isolation for molecular studies). Please visit www.lifetechnologies.com/cellisolation and follow our QuickLinks for recommended sample preparation procedures.

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.
Never use less than 25 μl (1 x 10⁷) Dynabeads per ml of cell sample and at least 4 Dynabeads per target cell.

Table 1: Volume of Dynabeads added per ml of cell sample. The volumes can be scaled up as required.

* if the concentration of MNC is increased, the volume of Dynabeads must be increased accordingly. Cell concentration can be up to 1 x 10⁸ cells per ml.


Depletion or Positive Isolation of CD15+ Cells

1. Add the appropriate volume of Dynabeads to the prepared sample according to table 1.
   
   
2. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.
   
   
3. Place the tube in a magnet for 2 min.
   
   
4. For depletion, transfer supernatant to a new tube for further use.
   
   
5. For positive isolation, discard the supernatant and wash the beadbound cells 3 times by resuspending in Buffer 1 to the original sample volume, and separate using a magnet. For molecular studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for protein or gene expression analysis.

1. Bettelheim P. (1989) Cluster report: CD15. In Leukocyte Typing IV (ed. W. Knapp et al.), p 798. Oxford University Press, Oxford.


2. Cao TM et al (2005) Engraftment and survival following reducedintensity allogeneic peripheral blood hematopoietic cell transplantation is affected by CD8+ T cell dose. Blood. 105:2300-2306


3. Beckhove P et al (2004) Specifically activated memory T cell subsets from cancer patients recognize and reject xenotransplanted autologous tumors. J. Clin. Invest. 114:67–76.


4. Smith D (2004) Liver Transplant- Associated Graft-versus-host Disease. ASHI Quarterly Q1: 12-13.