MagCellect Mouse Mesenchymal Stem Cell Isolation Kit

Assay Procedure

Refer to the product datasheet for complete product details.

View Graphic Procedure

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:

1. Soleimani, M. and S. Nadri (2009) Nature Protocols 4(1):102.
2. 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: