MagCellect Mouse Mesenchymal Stem Cell Isolation Kit
R&D Systems, part of Bio-Techne | Catalog # MAGM212B
Key Product Details
Summary for MagCellect Mouse Mesenchymal Stem Cell Isolation Kit
For the isolation of mouse mesenchymal stem cells (MSCs) using negative selection
Key Benefits
- Yields highly pure (75-95%) mouse MSCs in under an hour
- Negative selection principle yields an untouched MSC population
- Enrichment of MSCs reduces experimental variation
- Cost effective for the processing of multiple samples when used with the MagCellect™ Magnet from R&D Systems (Catalog # MAG997)
- Compatible with MidiMACS™ and EasySep® magnets and columns
Why Isolate Mesenchymal Stem Cells?
The term ‘mesenchymal stromal cells’ is commonly used to describe a heterogeneous population of cultured cells that are adherent to plastic, have a distinct morphology, and express a specific set of marker proteins. Within this heterogeneous population are cells referred to as ‘mesenchymal stem cells.’ Mesenchymal stem cells are multipotent, self-renewing cells that have the ability to differentiate into adipocytes, chondrocytes, and osteoblasts when cultured in vitro. The isolation of mesenchymal stem cells facilitates research into their multipotent properties and their role in regenerative medicine and cell therapy.
The MagCellect™ Mouse Mesenchymal Stem Cell Isolation Kit contains the following reagents that allow for isolation of highly pure mouse MSCs.
- Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail
- Streptavidin Ferrofluid
- Plus 10X Buffer
This kit contains sufficient reagents to process 300 x 106 cells; up to 12 isolations.
 | Enrichment of MSCs from C57/BL6 Mouse Compact Bone Using the MagCellect™ Mesenchymal Stem Cell Isolation Kit. Cells before (A) and after (B) mesenchymal stem cell enrichment were stained with Anti-Mouse PDGF Rα Alexa Fluor® 488-conjugated Antibody (R&D Systems, Catalog # FAB1062G) and an Anti-Mouse CD45-APC Monoclonal Antibody (R&D Systems, Catalog # FAB114A). (C) Morphology of isolated and unstained MSCs demonstrating a typical mesenchymal stem/stromal cell shape. |
The term 'mesenchymal stem cells' (MSCs) is most commonly used to describe multipotent self-renewing cells that can be differentiated in vitro to generate adipocytes, chondrocytes, and osteoblasts. However, because these biological properties and hierarchical relationships remain to be clearly demonstrated in vivo, the term 'multipotent mesenchymal stromal cells' is often used to distinguish cultured cells from their in vivo precursors. Originally discovered in mouse bone marrow, multipotent mesenchymal stromal cells cultured from a variety of species and tissue types, have been shown to differentiate into progeny of additional lineages including, cardiomyocytes, endothelial cells, hepatocytes, and neural cells. Again, the physiological relevance of these findings remains to be determined.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, mouse mesenchymal stem/stromal cells can be isolated using the following procedure:
- Incubate the single-cell suspension with the Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail
- Incubate the cells in the Streptavidin Ferrofluid
- Place the tube in a magnetic field
- Collect the desired cells while undesired cells remain attracted to the magnet
The MagCellect™ Mouse Mesenchymal Stem Cell Isolation Kit contains the following reagents that allow for isolation of highly pure mouse MSCs.
- Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail
- Streptavidin Ferrofluid
- Plus 10X Buffer
This kit contains sufficient reagents to process 300 x 106 cells; up to 12 isolations.
Reagents
- MagCellect™ Magnet (Catalog # MAG997), or equivalent.
This kit is compatible with MidiMACS™ and EasySep® magnets and columns. - 12 x 75 mm (5mL) polystyrene round bottom tubes
- Sterile Pasteur pipettes or transfer pipettes
- Mouse Erythrocyte Lysing Kit (R&D System, Catalog # WL2000).
Precaution:
Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
Cell Preparation
Use preferred or traditional methods to prepare a single cell suspension from mouse bone marrow or compact bone. For a general protocol to isolate compact bone and bone marrow cells from mice, please refer to:
- Soleimani, M. and S. Nadri (2009) Nature Protocols 4(1):102.
- Short, B.J. et al. (2009) Methods Mol. Biol. 482:259.
This procedure is for the processing of 25 x 106 total cells using 5 mL tubes and the MagCellect™ Magnet. It is not recommended to use less than 10 x 106 total cells. Cells and reagents should be kept cold using an ice bath or a refrigerator. Reaction incubations must be carried out at 2-8 °C in a refrigerator and not in an ice bath to avoid excessively low temperatures that can slow the kinetics of the optimized reactions.
Generate a single cell suspension in HBSS (or equivalent) supplemented with 10% bovine serum.
Filter the suspended cells through a 40-70 µm nylon cell strainer.
Wash the cells once with HBSS containing 10% bovine serum.
Centrifuge the cells at 200 x g for 10 minutes.
Decant the supernatant.
Remove red blood cells if necessary using R&D Systems® Mouse Erythrocyte Lysing Kit (Catalog # WL2000).
Resuspend the cells in cold 1X Plus Buffer.
Perform a cell count.
Adjust the cell concentration to 5 x 107 cells/mL with cold 1X Plus Buffer.
Transfer 25 x 106 cells (0.5 mL) into a 5 mL polystyrene tube.
Add 20 µL of Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail.
Mix the cell-antibody suspension gently.
Incubate at 2 °C to 8 °C for 15 minutes.
Wash cells with 1X Plus Buffer, centrifuge the cells at 200 x g for 10 minutes, and resuspend cells in 1 mL of 1X Plus Buffer.
Add 250 µL of Streptavidin Ferrofluid to the cell suspension.
Mix gently.
Incubate at 2 °C to 8 °C for 15 minutes.
Add 1.35 mL of 1X Plus Buffer.
Mix gently.
Place the reaction tube in the MagCellect™ Magnet.
Incubate at room temperature for 6 minutes to remove undesired cells. Magnetically tagged cells will migrate toward the magnet (these are the unwanted cells), leaving the mouse mesenchymal stem cells in suspension in the supernatant.
Transfer the supernatant containing the mouse MSCs into a new 5 mL tube.
Repeat the magnetic depletion with the new tube containing the recovered cells. The supernatant obtained at the end of these steps is the final depleted cell fraction containing the desired enriched mesenchymal stem cells.
Cells are now ready for counting and further downstream applications. The mouse MSCs isolated by negative selection typically have a purity of 75-90%.
Technical Hints
- If sterile cells are required following the cell selection, the entire procedure should be carried out in a laminar flow hood to maintain sterile conditions. Use sterile equipment when pipetting reagents that will be reused at a later date.
- Avoid antibody capping on cell surfaces and non-specific cell tagging by working efficiently, keeping cells and solutions cold through the use of pre-cooled solutions and by adhering to the incubation times and temperatures specified in the protocol. Increased temperature and prolonged incubation times may lead to non-specific cell labeling thus lowering cell purity and yield.
- Use the following table for recommended quantities to be used in during the cell isolation procedure:
| Number of Cells in Starting Preparation | 25 x 106 | 50 x 106 |
|---|---|---|
| Reaction Volume | 0.5 mL | 0.5 mL |
| Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail | 20 µL | 40 µL |
| Streptavidin Ferrofluid | 250 µL | 400 µL |
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