Introduction

Dynabeads Sheep anti-Mouse IgG in combination with primary mouse antibodies are ideal for depletion or positive isolation of cells from different species (e.g. human), 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.

The polyclonal sheep anti-mouse IgG antibodies bind both heavy and light chains of mouse IgG1, IgG2a and IgG2b. They will also bind to light chains of mouse immunoglobulins of all isotypes (e.g. IgG3 and IgM) but with a lower reactivity.

Note:
   Dynabeads ClinExVivo™ Sheep anti-Mouse IgG (Cat. no. 422.01) are available for ex vivo separation of human cells for cell-based clinical research.

Principle of Isolation

The primary mouse 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 Sheep anti-Mouse IgG are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with polyclonal Sheep anti-Mouse IgG antibodies. The polyclonal antibodies have been affinity adsorbed to reduce cross-reactivity to human immunoglobulins. Cross-reactivity to rat antibodies is high.

Materials Supplied

5 ml Dynabeads Sheep 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

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

Ordering Information

Sku Name Size Price Qty
11031 Dynabeads® Sheep-Anti Mouse IgG 5 mL USD 626.00

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  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 volume of Dynabeads must be increased accordingly. Cell concentration can be up to 1 x 108 cells per ml.

Cell Isolation - Indirect Technique

Labeling Cells with Mouse IgM 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 0.5 – 1.5 μg of primary antibody (mouse IgG) per 25 μl (1 x 107) Dynabeads. It is necessary to titrate the amount of antibody.

 

  1. Transfer washed Dynabeads to a tube.

  2. Add antibodies.

  3. Incubate for ≥ 30 minutes at 2-8°C 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 Dynabeads in the same volume Buffer 1 as the initial volume of Dynabeads.


Isolation or Depletion of 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 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 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.


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.
TOP

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 .
TOP

References

  1. Anjuère F et al. (2004) In vivo adjuvant-induce mobilization and maturation of gut dendritic cells after oral administration of Cholera toxin. J. Immunol. 173: 5103-5111.

  2. Chai JG et al. (2000) CD152 ligation by CD80 on T cells is required for the induction of unresponsiveness by costimulation deficient antigen presentation. J. Immunol. 165: 3037-3042.

  3. Delneste Y et al. (2003) Interferon-g switches monocyte differentiation from dendritic cells to macrophages. Blood 101: 143-150.

  4. Gutiérrez-Frías C et al. (2004) Sonic hedgehog regulates early human thymocytes differentiation by counteracting the IL-7-induced development of CD34+ precursor cells. J. Immunol. 173: 5046-5053.

  5. Hall A et al. (2004) Rh Autoantigen presentation to helper T cells in chronic lymphocytic leukemia by malignant B-cells. Blood. Jul 29 [Epub ahead of print].

  6. Ho LH et al. (2004) Labile zinc and zinc transporter ZnT4 in mast cell granules: Role in regulation of caspas  activation and NF-kB translocation. J. Immunol. 172: 7750-7760.

  7. Inobe M et al. (2004) CTLA-4 engagement acts as a brake on CD4+ T cell proliferation and cytokine production but is not required for tuning T cell reactivity in adaptive tolerance. J. Immunol. 173: 7239-7248.

  8. Jaleco S et al. (2003) Homeostasis of naïve and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis. J.Immunol. 171: 61-68.

  9. Kawamura T et al. (2003) Decreased stimulation of CD4+ T cell proliferation and IL-2 production by highly enriched populations of HIV-infected dendritic cells. J. Immunol. 170: 4260- 4266.

  10. Liu DY et al. (2002) An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm. Mol. Human Reproduction. 8(1): 37-47.

  11. Mackay G et al. (2004) Protection against lateonset AIDS in macaques prophylactically immunized with a live simian HIV vaccine was dependent on persistence of the vaccine virus. J. Immunol. 173: 4100-4107.

  12. Pater-Huijsen FL et al. (2002) Products from human mast cell lines cells enhance the production of interferon-gamma by CD8(+) and CD4(+) T cells. Immunology 106(1):11-19.

  13. Pollara G et al. (2004) Herpes Simplex virus type-1 induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion. J. Immunol. 173: 4108-4119.

  14. Sompuram SR et al. (2002) Synthetic peptides identified from phage-displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets. Clin. Chemistry 48(3): 410-420.

  15. Strom TS et al. (2003) Defects in T cell-mediated immunity to influenza virus in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene transfer into repopulating hematopoietic cells. Blood 102: 3108-3116.

  16. Wang JCE et al. (2003) Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo. J. Immunol. 171: 6339-6343.

  17. Weissman D et al. (2000) HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response. J. Immunol. 165: 4710-4717.

  18. Xystrakis E et al. (2004) Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat. J. Immunol. 173: 3140-3147.

  19. Martinez del Hoyo G et al. (2006) Prion Protein Expression by Mouse Dendritic Cells Is Restricted to the Nonplasmacytoid Subsets and Correlates With the Maturation State. J Immunol. 177:6137-6142.

  20. Shannon-Lowe C et al. (2005) Epstein-Barr virus- induced B-cell transformation: quantitating events from virus binding to cell outgrowth. J Gen Virol.56:3009-3019.

  21. Omidvar N et al. (2006) Expression of Glycosylphosphatidylinositol- Anchored CD59 on Target Cells Enhances Human NK Cell-Mediated Cytotoxicity. J Immunol. 176:2915-2923.

  22. Grigoriadis A et al. (2006) Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data. Breast Cancer Res. 8:R56.

  23. Ohyama M et al. (2006) Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest.116:249-260.

 

TOP
110.31.indd   Rev 002    May-5-2008