Goat Anti-Mouse IgG - Depletion or Positive Isolation of Cells from Different Species

Dynabeads Goat anti-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.

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 bead-bound 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 Goat anti-Mouse IgG are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with polyclonal goat anti-mouse IgG antibodies. The antibody coated onto Dynabeads recognises the heavy chain of most mouse IgG subclasses and is Fc-reactive. Cross-reactivity to human antibodies is minimal.

Materials Supplied

• 5 mL Dynabeads Goat anti-Mouse IgG 
• 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 10 ⁹ cells

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

• Mouse IgG antibodies.
• Magnet: (Dynal MPC™) - MPC-S for 20 μl to 2 mL samples, MPC-L for 1-8 mL samples, MPC-15 for 1-15 mL samples and MPC-50 for 15-50 mL samples.
• Mixer allowing both tilting and rotation.
• Buffer 1: PBS (without Ca²⁺ and Mg²⁺) w/0.1% BSA and 2 mM EDTA, pH 7.4.

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/samplepreparation 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.

* 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 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:
               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.

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 mouse IgG 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:
⁷



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

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 Mouse IgM Antibodies

• 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.

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 .

1. Abe M et al (2004) Osteoclasts enhance myeloma cell growth and survival via cell-cell contact: a vicious cycle between bone destruction and myeloma expansion. Blood 104: 2484-2491.
2. Bas A et al (2003) Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression. J. Immunol. 171: 3359-3371.
3. Cassoni P et al (2002) Oxytocin is a growth factor for Kaposi’s sarcoma cells: Evidence of endocrine-immunological cross-talk. Cancer Research 62(8): 2406-2413.
4. Das H et al (2004) Mechanisms of Vd1 gd T cell activation by microbial components. J. Immunol. 172: 6578-6586.
5. Gilliet M et al (2003) Human dendritic cells activated by TSLP and CD40L induce proallergic cytotoxic T cells. J. Exp. Med. 197:1059-63.
6. Curnow SJ et al (2004) Topical glucocorticoid therapy directly induces up-regulation of functional CXCR4 on primed T lymphocytes in the aqueous humor of patients with uveitis. J. Immunol. 172: 7154-7161.
7. Kodali S et al (2004) Vasoactive intestinal peptide modulates Langerhans cell immune function. J. Immunol. 173: 6082-6088.
8. Lee JC et al (2004) Elevated TGF-b1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. J. Immunol. 172: 7335-7340.
9. Liau LM et al (2002) Tumor immunity within the central nervous system stimulated by recombinant Listeria monocytogenes vaccination. Cancer Research 62(8), 2287-2293.
10. Lozupone F et al (2004) Effect of human natural killer and gd T cells on the growth of human autologous melanoma xenografts in SCID mice. Cancer Res. 64: 378-385.
11. Manna PP et al (2003) The mechanism of CD47-dependent killing of T cells: heterotrimeric Gi-dependent inhibition of Protein Kinase A. J. Immunol. 170: 3544-3553.
12. Shuford WW et al (1997) 4-1BB Co-stimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186: 47-55.
13. Vahlenkamp TW et al (2004) Feline immunodeficiency virus infection phenotypically and functionally activate  immunosuppressive CD4+CD25+ T regulatory cells. J.Immunol. 172:4752-61.
14. von Essen M et al (2004) Constitutive and ligand-induced TCR degradation. J. Immunol. 173: 384-393.
15. Zambello R et al (2003) Expression and function of KIR and natural cytotoxicity receptors in NK-type lymphoproliferative diseases of granular lymphocytes. Blood. 102: 1797-1805.
16. Zingoni A et al (2004) Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions. J. Immunol. 173:3716-3724.