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

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:

• 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, pH 7.4.
• Mouse monoclonal antibodies.

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.

*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. 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.

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.

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