Introduction

Dynabeads Pan Mouse IgG in combination with primary mouse IgG 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.

Note:  For positive isolation of cells for downstream cellular applications (bead-free cells), please use the equivalent CELLection™ Pan Mouse IgG product.

Principle of Isolation

The primary mouse IgG 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 Pan Mouse IgG are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal human anti-mouse IgG antibody. The antibody coated onto Dynabeads recognizes all mouse IgG subclasses and is Fc specific. The Pan Mouse IgG antibody does not crossreact with human, rat, rabbit, guinea pig, sheep, goat or hamster IgG.

Materials Supplied

Dynabeads Pan Mouse IgG Cat. no. Volume
4 x 108 Dynabeads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3). 110.41 5 ml
  110.42 5 x 5 ml

Each 5 ml of product can be used to process up to 2 x 109 cells

Additional Materials Required

 

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).


Sample Preparation


Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC suspensions 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
1 ml 
Volume of Dynabeads 25 μl 50 μl
Total no. of cells
processed per product
2 x 109 cells
1 x 109 cells


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

Cell Isolation - Indirect Technique

Labeling Cells with Mouse IgG Antibodies

  • Use approximately 1 μg of primary antibody (mouse IgG) per 106 target cells.
  • Recommended cell concentration: 1 x 107 cells/ml.
  1. Add primary antibody to the cell suspension and mix.

  2. Incubate for 10 min at 2-8°C.

  3. 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.

  4. Resuspend the cells in Buffer 1 at 1 x 107 cells per ml.

  5. Proceed to Isolation or Depletion of Cells


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


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



Cell Isolation - Direct Technique


Pre-coating Dynabeads

  • Use 0.2 – 0.5 μg of primary antibody (mouse IgG) per 25 μl (1 x 107) Dynabeads.

 

  1. Transfer washed Dynabeads to a tube.

  2. Add mouse IgG 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 as 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 application.

Technical Recommendations

Indirect versus Direct Technique

Use the indirect technique when:

  • A cocktail of mouse monoclonal antibodies is used.
  • Very high depletion efficiency is needed.
  • The affinities of mouse monoclonal 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 Mouse IgG Antibodies

  • Titrate the primary antibody to optimize the amount used.
  • To avoid non-specific binding of cells (e.g. monocytes, B cells), add aggregated IgG to block Fc receptors prior to adding the primary antibodies.
  • 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 IgG 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

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  2. Berlyn ICA et al. (2001) Generation of CD4+ and CD8+ T lymphocyte responses by dendritic cells armed with PSA/anti-PSA antigen/antibody) complexes. Clin. Immunol. 101(3): 276:283.

  3. Hofmann I et al. (2002) Symplekin, a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes. Mol. Biol. Cell. 13(5): 1665-1676.

  4. Högerkorp CM et al. (2003) CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analysed by DNA microarrays. Blood 101: 2307-2313.

  5. Lee E et al. (2004) Increased expression of Interleukin 23 p19 and p40 in lesional skin of patients with Psoriasis Vulgaris. J. Exp. Med. 199(1): 125-130.

  6. Llewelyn M et al. (2004) HLA Class II polymorphisms determine responses to bacterial superantigens. J. Immunol. 172: 1719-1726.

  7. Morelli A et al. (2004) Endocytosis, intracellular sorting and processing of exosomes by dendritic cells. Blood 104: 3257-3266.

  8. Saemann MD et al. (2002) Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells. J. Leukocyte Biol. 71(2): 238-246.

  9. Tiwari A et al. (2003) Extraction of cells for single-stage seeding of vascular-bypass grafts. Biotechnol. Appl. Biochem. 38: 35-41.

  10. Woywodt A et al. (2004) Counting the cost: markers of endothelial damage in hematopoietic stem cell transplantation. Bone Marrow Transplantation 34: 1015-1023.

  11. Woywodt A et al. (2003) An improved assay for enumeration of circulating endothelial cells. Ann. Hematology 83(8): 491-494.
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110.41.42.indd  Rev 002    May-5-2007