MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit

Assay Procedure

Refer to the product datasheet for complete product details.

View Graphic Procedure

Reagents Supplied in the MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit (Catalog # MAGM209)

• MagCellect Mouse Cell Lineage Depletion Biotinylated Antibody Cocktail; 1 mL in PBS with BSA
• MagCellect Streptavidin Ferrofluid; 2 mL in solution containing BSA and preservatives
• MagCellect Blocking Reagent; 0.5 mL rat IgG in PBS with BSA
• MagCellect Plus Buffer (10X) – 25 mL of 10X concentrate

This kit contains sufficient reagents to process up to 1 x 10⁹ total cells.

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

Generate a mononuclear suspension in cold 2% FBS-PBS.

Wash the cells once with Hank’s BSS containing 10% bovine serum.

Centrifuge the cells at 300 x g for 8 minutes.

Decant the supernatant.

Resuspend the cells in cold 1X MagCellect Plus Buffer.

Perform a cell count.

Transfer 1 x 10⁸ cells (5 mL) into a 15 mL centrifuge tube.

Add 50 µL of MagCellect Blocking Reagent.

Incubate at 2 ^°C to 8 ^°C for 15 minutes.

Add 100 µL of MagCellect Mouse Cell Lineage Depletion Biotinylated Antibody Cocktail.

Mix the cell-antibody suspension gently.

Incubate at 2 ^°C to 8 ^°C for 15 minutes.

Centrifuge the cells at 300 x g for 8 minutes.

Resuspend the cells in 1 mL of cold 1X MagCellect Buffer.

Add 150 µL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Mix gently.

Incubate at 2 ^°C to 8 ^°C for 15 minutes.

Add 0.85 mL of 1X MagCellect 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 lineage-negative cells in suspension in the supernatant.

Transfer the supernatant containing the mouse lineage-negative cells 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 lineage-negative hematopoietic cells.

The cells are now ready for counting and further downstream applications. The resulting cell population is typically highly enriched for CD117+ cells (40-70%) with less than 5% residual lineage-positive cells.

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.
• When processing different numbers of cells observe the following guidelines: keep blocking, antibody cocktail and ferrofluid incubation times and temperatures the same. Add 5 μL of Blocking Reagent-1 per each 1 x 10⁷ cells being processed. Add 10 μL of Antibody Cocktail per each 1 x 10⁷ cells being processed. Add 15 μL of Streptavidin Ferrofluid per each 1 x 10⁷ cells being processed.
• When processing 2 x 10⁸ cells or fewer use the 12 x 75 mm (5 mL) tubes with the MagCellect Magnet horizontally positioned to accommodate up to six 5 mL tubes. Do not process more than 2 x 10⁸ cells in each 5 mL tube and do not exceed a total reaction volume of 3 mL in each tube. A reaction volume of 2 mL is recommended for processing 1 x 10⁸ cells. A reaction volume of 1 mL is recommended when processing 5 x 10⁷ or fewer cells. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.
• When processing greater than 2 x 10⁸ cells use the 17 x 100 mm (15 mL) tubes with the MagCellect magnet positioned vertically to accommodate up to two 15 mL tubes. Do not process more than 6 x 10⁸ cells in each 15 mL tube and do not exceed a total reaction volume of 9 mL in each tube. When using this larger tube, increase the reaction volume before the magnetic separation step according to the following formula: 3 mL for each 2 x 10⁸ cells processed. Also increase the incubation time in the magnet described to 8 minutes. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.

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