Introduction

Dynabeads Rat anti-Mouse IgM in combination with primary mouse IgM antibodies are ideal for depletion or positive isolation of cells from different species (e.g. human, rat) depending on the specificity of the primary antibody. Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC suspensions or tissue digests.

Principle of Isolation
The primary mouse IgM antibody is either added to the cell sample (indirect technique) or pre-coated onto the beads (direct technique) prior to cell isolation.

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

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

Description of Materials
Dynabeads Rat anti-Mouse IgM are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated wit  mouse IgM antibodies. The antibody coated onto Dynabeads recognises the μ heavy chain of mouse immunoglobulins. No cross-reactivity to rabbit, swine or human IgG. Low crossreactivity to chicken, goat, sheep, bovine, horse, dog, baboon IgG and human IgM.

Materials Supplied

  • 5 ml Dynabeads Rat anti-Mouse IgM
  • 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

  • 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.
  • Mouse IgM antibodies.

Important Notes:

  • BSA can be replaced by human serum albumin (HSA) or FCS.
  • EDTA can be replaced by 0.6% sodium citrate.
  • PBS containing Ca2+ or Mg2+ is not recommended.

Ordering Information

Catalog # Name Size List Price (USD) Qty
11039D Dynabeads® Rat Anti-Mouse IgM 5 mL 596.00

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 a  the initial volume of Dynabeads (step 2).


Sample Preparation

Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC or tissue digests. 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 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 cells 1 ml Max 2.5 x 106 target cells
Volume of Dynabeads 25 μl 50 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 - Indirect Technique

Labelling Cells with Mouse IgM Antibodies

  • Use approximately 1 mg antibody per 106 target cells.
  • Recommended cell concentration: 1 x 107 cells/ml.
  1. Add mouse IgM antibodies to the cell suspension. Mix well and incubate for 20 min at 2-8°C.
  2. Wash the cells by adding 2 ml Buffer 1 per 1 x 107 cells and centrifuge at 300 x g for 8 min. Discard the supernatant.
  3. Resuspend the cells in Buffer 1 at 1 x 107 cells/ml.


Isolation or Depletion of Cells

  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. Double the volume with Buffer 1 to limit trapping of unbound cells (optional).
  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



Cell Isolation - Direct Technique

Pre-coating Dynabeads

Use 0.5–1.5 μg mouse IgM antibodies per 25 μl (1 x 107) Dynabeads. 

  1. Transfer washed Dynabeads to a tube.
  2. Add antibodies.
  3. Incubate for ≥ 30 min with gentle tilting and rotation.
  4. Place the tube in a magnet for 1 min and discard the supernatant.
  5. Wash the beads twice using 2 ml of Buffer 1.
  6. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 a  the initial volume of Dynabeads.


Isolation or Depletion of Target Cells

  1. Add the pre-coated Dynabeads to the cells 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. Double the volume with Buffer 1 to limit trapping of unbound cells (optional).
  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:

            i) Add 1 ml Buffer 1 per 1 x 107 Dynabeads.
            ii) Place the tube in the magnet for 1 min and discard the supernatant.

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

Technical Recommendations

Indirect versus Direct Technique

Use the indirect technique when:

  • A cocktail of mouse IgM antibodies is used.
  • Very high depletion efficiency is needed.
  • The affinities of mouse IgM antibodies are low.
  • The cells express low number of target antigens.
  • The direct technique gives unsatisfactory purity.


The direct technique may be used if:

  • The affinity of the primary antibody is high.
  • The cells express a high number of target antigens.
  • A stock preparation of primary coated Dynabeads is desired.


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 onto beads (direct technique), even though the antibody shows good results in other immunological assays.

Labeling Cells with IgM Antibodies

  • Titrate the primary antibody to optimise the amount used.
  • To avoid aggregation of cells it is recommended to use gamma-globulin or Fc blocking reagents.
  • Excess antibody must be removed by washing before cell isolation.


Isolation and Depletion of Target Cells

  • Remove density gradient media (e.g. Ficoll): Wash cells prior to adding mouse IgM 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.
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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

  1. Estes JD et al (2004) Follicular Dendritic Cell Regulation of CXCR4-Mediated Germinal Center CD4 T Cell Migration1. J. Immunol. 173: 6169– 6178.
  2. Nikolov EN and Ivanova-Nikolova T (2004) Coordination of Membrane Excitability through a GIRK1 Signaling Complex in the Atria. J. Biol.Chem. 279 (22): 23630–23636.
  3. Test ST et al (2005) Depletion of Complement Has Distinct Effects on the Primary and Secondary Antibody Responses to a Conjugate of Pneumococcal Serotype 14 Capsular Polysaccharide and a T-Cell-Dependent Protein Carrier. Infection and Immunity, 73: 277–286.
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110.39D.indd   Rev 002    5-May-2007