Protocols

Introduction

Intended Use

Dynabeads M-450 Tosylactivated coupled with antibodies or other ligands provide a versatile tool for isolation of both cells and non-cell targets (e.g. proteins and other biomolecules). Their size makes them particularly suitable for stimulation and expansion of e.g. T cells (2,3,4,7). Cells can be directly isolated from any sample such as whole blood, bone marrow, mononuclear cell suspensions (MNC) or tissue digests. Note: For protein purification and immunoassays Dynabeads M-280 Tosylactivated (Cat. no.142.03/04), or Dynabeads MyOne™ Tosylactivated (Cat. no. 655.01/02/03) are recommended.


Principle of Coupling

Dynabeads M-450 Tosylactivated provide reactive sulphonyl esters that can react covalently with proteins (e.g. antibodies) or other ligands containing primary amino or sulphydryl groups. No further activation is necessary. Dynabeads M-450 Tosylactivated will bind proteins physically and chemically with an increasing number of covalent bonds with higher temperature and pH.

Principle of Isolation

Once coupled with the specific ligand, the Dynabeads are mixed with the sample in a tube. The Dynabeads will bind to the target during a short incubation, and then the bead-bound target is separated by a magnet.

  • Positive isolation – discard the supernatant and use the bead-bound target for downstream applications.
  • Depletion – discard the bead-bound target and use the remaining, untouched sample for downstream applications.

Description of Materials

Dynabeads M-450 Tosylactivated are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) with a surface suitable for physical and chemical binding of antibodies and other biomolecules.

Materials Supplied

5 ml Dynabeads M-450 Tosylactivated. 4 x 10 8 beads/ml supplied in distilled water.

Additional Materials Required

  • Magnet: (Dynal MPC™): See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.
  • Mixer allowing both tilting and rotation.
  • Buffer 1: 0.1 M sodium phosphate buffer, pH 7.4-8.0 or 0.1 M sodium borate buffer, pH 7.6- 9.5.
  • Buffer 2: Phosphate Buffered Saline (PBS) (without Ca2+ and Mg2+), 0.1% Bovine Serum Albumin (BSA) (w/v) and 2 mM EDTA, pH 7.4.
  • Buffer 3: 0.2 M Tris w/0.1% BSA, pH 8.5.
  • Optional: Sodium azide (NaN3) as preservative.
  • Blocking (optional): BSA.
  • Specific ligands.

Important Notes:

BSA can be replaced by human serum albumin (HSA).
EDTA can be replaced by sodium citrate.
PBS containing Ca 2+ or Mg 2+ is not recommended.

TOP

Protocols

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).


Critical Steps for Coupling of Ligands to Dynabeads

Buffers

  • Sugars and stabilisers may interfere with binding and should be removed from the ligand preparation.
  • Coupling buffers should not contain any reactive groups (amines, thiols and hydroxyls) e.g. tris, glycine or proteins.
  • The pH and the ionic strength of the coupling buffer can be varied, but generally the coupling reaction is carried out in 0.1 M phosphate buffer at pH 7.4-8.0. Tosyl groups are more reactive at higher pH; therefore 0.1 M borate buffer pH 7.6-9.5 can be used depending on the ligand stability. Buffers with higher ionic strength stimulate hydrophobic interactions, which facilitate the coupling efficiency.


Ligand and Dynabeads concentration

  • For cell separation purposes couple with 3-5 μg purified ligand per 25 μl (1 x 107) Dynabeads. Optimization of amount of ligand is recommended.
  • The Dynabeads concentration during coupling should be 4-8 x 108 Dynabeads per ml.


Coupling conditions

The physical adsorption of the ligand to the bead surface is rapid, while the formation of covalent bonds will need more time. After the recommended 16-24 hours at 37˚C, a maximal chemical binding is achieved. Coupling at 18-25˚C (RT) will require an extended incubation time to 48 hours and longer to obtain the same degree of chemical binding. The upper temperature is limited by the stability of the ligand.

Blocking

  • Adding a blocking protein such as 0.01-0.5% w/v BSA to the coupling solution may increase functionality of the coupled antibodies in cell isolation protocols. Blocking protein is generally added to coupling solution after 0-30 min. Both the incubation time before blocking and blocking protein concentration should be optimized.
  • For protein isolation applications, blocking may not be required.


Coupling of Ligands to Dynabeads

The following procedure is a suggested, general procedure for antibody coupling to Dynabeads. It can be scaled-up and optimized.
Use approximately 5 μg antibody per 25 μl (1 x 107) Dynabeads.
This procedure describes coupling to 1 ml (4 x 108) Dynabeads.

  1. Transfer 1 ml of washed Dynabeads to a tube.

  2. Place the tube in a magnet for 1 min and discard the supernatant. Remove the tube from the magnet.

  3. Resuspend Dynabeads in Buffer 1 (1 ml minus antibody volume) and add 200 μg antibody during mixing to reach a total coupling volume of 1 ml.

  4. Optional: Incubate for 15 min and then add BSA to 0.1% w/v.

  5. Incubate for 16-24 hours at 37°C with gentle tilting and rotation.

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

  7. Wash the coated beads 3-4 times:

    a.   Wash twice for 5 min at 2 - 8°C with 1 ml Buffer 2.

    b.   Optional: incubation for 24 hours at RT or 4 hours at 37°C in Buffer 3 will deactivate remaining free tosyl groups.

    c.   Wash once for 5 min at 2 - 8°C in 1 ml Buffer 2.

  8. Remove the tube from the magnet and resuspend the Dynabeads in 1 ml Buffer 2 (to obtain 4 x 108 beads/ml). The beads are coated and ready for use.


Sample Preparation

Cells can be directly isolated from any sample such as whole blood, bone marrow, mononuclear cell suspensions (MNC) or tissue digests. Please visit www.dynalbiotech.com/samplepreparation for a list of 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 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.


 Positive Isolation Depletion
Sample volume (1 x 107 cells/ml*)1 ml Max 2.5 x 106 target cells1 ml Max 2.5 x 106 target cells
Volume of Dynabeads25 μl50 μl


* If the concentration of cells is increased or the target cell concentration exceeds 2.5 x 106, the Dynabeads volume must be increased accordingly. Cell concentration can be up to 1 x 108 cells per ml.


Cell Isolation

  • Wash coupled Dynabeads before use to remove any soluble ligand. Use the Dynabeads Washing Procedure  replacing Buffer 1 with Buffer 2.

 

  1. Add 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. Optional: Double the volume with Buffer 2 to limit trapping of unbound cells.

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

  5. Depletion: Transfer the supernatant containing the unbound cells to a fresh tube for further experiments.

  6. Positive isolation: Discard the supernatant and gently wash the bead-bound cells 4 times, using the following procedure:

    7. 7



  7. Resuspend the cells in buffer/medium for downstream application.

 

Technical Advice

Antibody Selection

The choice of primary antibody is the most important factor for successful cell isolation. Note that some antibodies may show reduced antigen-binding efficiency when coated directly onto beads, even if the antibody shows good results in other immunological assays. See section Spacer and Ligand Orientation for recommendation of our secondary coated products.

Spacer and Ligand Orientation

The efficiency of antibody-antigen binding can be increased for some applications by using a spacer molecule e.g. a secondary antibody coupled to the Dynabeads prior to coupling of the primary antibody. Invitrogen Dynal offers several secondary coated Dynabeads for cell isolation. For further information, please visit www.invitrogen.com.

Storage of Coupled Dynabeads

  • Store coupled Dynabeads in Buffer 2 at 2-8°C in liquid suspension. Coupled Dynabeads may typically be stored for months or even years. Stability of coupled Dynabeads depends on ligand stability and must be determined separately.
  • A final concentration of 0.02% NaN3 can be added as a preservative to the storage buffer. The preservative must be removed by washing prior to cell isolation.


Isolation and Depletion of Target Cells

Remove density gradient media (e.g. Ficoll): Wash cells prior to adding antibodies or Dynabeads.
Remove soluble factors in serum: Serum may contain soluble factors (e.g. antibodies or cell surface antigens), which can interfere with the cell isolation protocol. Washing the cells once may reduce this interference.

References

  1. Clavet CR et al (2004) Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts. J. Virol. Methods 119(2): 121-8.

  2. Erdmann A et al (2005) Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2 driven expansion in vivo. Blood. Mar. 3 (E-pub ahead of print).

  3. Hamad ARA et al (2003) B220+ double-negative T cells suppress polyclonal T cell activation by a Fas-independent mechanism that involves inhibition of Il-2 production. J. Immunol. 171: 2421-2426.

  4. Jung U et al (2003) CD3/CD28-costimulated T1 and T2 subsets: differential in vivo allosensitization generates distinct GVT and GVHD effects. Blood. 102: 3439-3446.

  5. Takemoto M et al (2002) A new method for large-scale isolation of kidney glomeruli from mice. Am. J.Pathol. 161: 799-805.

  6. Tenedini E et al (2004) Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells. Blood. 104: 3126-3135.

  7. Viglietta V et al (2004) Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. 199: 971-979.

  8. Wen J et al (2005) Use of superparamagnetic microbeads in tracking subretinal injections. Molecular Vision 11:256-62.
140.13.indd     Rev 002     5-May-2007