Dynabeads® M-450 Tosylactivated

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.

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

• 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.

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