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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 bead-bound cells are separated by a magnet.
Description of Materials
Dynabeads Rat anti-Mouse IgM are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with 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 cross-reactivity to chicken, goat, sheep, bovine, horse, dog, baboon IgG, and human IgM.
Materials Supplied
This product will process up to 2 x 109 cells.
Additional Materials Required
Important Notes:
Dynabeads Washing Procedure
Dynabeads should be washed before use.
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.
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
Labeling Cells with Mouse IgM Antibodies
Isolation or Depletion of Cells
Cell Isolation - Direct Technique
Pre-coating Dynabeads
Use 0.5–1.5 μg mouse IgM antibodies per 25 μl (1 x 107) Dynabeads.
Isolation or Depletion of Target Cells
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.
Indirect versus Direct Technique
Use the indirect technique when:
The direct technique may be used if:
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
Isolation and Depletion of Target Cells
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 .
For Research Use Only. Not for use in diagnostic procedures.