MagCellect Mouse Mesenchymal Stem Cell Isolation Kit

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 MagCellect Antibody Cocktail
• Add the MagCellect Streptavidin Ferrofluid
• Place the tube in a magnetic field
• Collect the desired cells while undesired cells remain attracted to the magnet

View Graphic Procedure

 

Reagents Supplied in the MagCellect Mouse Mesenchymal Stem Cell Isolation Kit (Catalog # MAGM212):

• MagCellect Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail; 0.25 mL in PBS with BSA
• MagCellect Mouse Streptavidin Ferrofluid; 3.0 mL in solution containing PBS with BSA and preservatives
• MagCellect Plus Buffer (10X) – 10 mL of 10X concentrate

This kit contains sufficient reagents to process 300 x 10⁶ total cells; up to 12 isolations.

• MagCellect Magnet (Catalog # MAG997), or equivalent
• 12 x 75 mm (5mL) polystyrene round bottom tubes
• Sterile Pasteur pipettes or transfer pipettes

1. Generate a single cell suspension in Hanks’ BSS (or equivalent) supplemented with 10% bovine serum.
2. Wash the cells once with Hank’s BSS containing 10% bovine serum.
3. Centrifuge the cells at 200 x g for 10 minutes.
 

4. Decant the supernatant.
5. Remove red blood cells if necessary using R&D Systems Mouse Erythrocyte Lysing Kit (Catalog # WL2000).
6. Resuspend the cells in cold 1X MagCellect Plus Buffer.
 

7. Perform a cell count.
8. Adjust the cell concentration to 5 x 10⁷ cells/mL with cold 1X MagCellect Plus Buffer.
 

9. Transfer 2.5 x 10⁷ cells (0.5 mL) into a 5 mL polystyrene tube.
10. Add 20 μL of MagCellect Mouse Mesenchymal Stem Cell Biotinylated Antibody Cocktail.
11. Mix the cell-antibody suspension gently.
12. Incubate at 2 °C to 8 °C for 15 minutes.
 

13. Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.
14. Mix gently.
15. Incubate at 2 °C to 8 °C for 15 minutes.
 

16. Add 1.35 mL of 1X MagCellect Plus Buffer.
17. Mix gently.
 

18. Place the reaction tube in the MagCellect Magnet.
19. 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.
 

20. Transfer the supernatant containing the mouse MSCs into a new 5 mL tube.
21. 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.
 

22. The cells are now ready for counting and further downstream applications. The mouse MSCs isolated by negative selection typically have a purity of 90-98%.
 

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 fast, 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 labelling thus lowering cell purity and yield.
• If very high levels of purity of the isolated mesenchymal stem cells are needed, consider performing a positive selection using Mouse Sca-1⁺ Stem Cells PlusCellect kit (Catalog # PLS1226) after using the MagCellect Mouse Mesenchymal Stem/Stromal Cell Isolation Kit.

Most difficulties arise from the following areas:

• Quality of the Cell Preparation: Dead cells and debris might interfere with isolation and detection, affecting both the purity and recovery of intended cells. Cell aggregation might also affect the enrichment performance.
• Few Expected Target Cells: If less than 100,000 expected target cells are present, recovery and/or purity of the isolation is compromised.