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MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit

Key Product Details

Enrichment of SCF R/c-kit in Bone Marrow Lineage-negative Cells.
(2)
Depletion of Lineage-committed Hematopoietic Cells.
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MAGM209

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mouse lineage-negative hematopoietic 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
 

 

Kit Components

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; 1.5 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.

 

 

Other Supplies Required
  • 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
 

 

Procedure Overview

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.

Enrich for mononuclear cells by using a density gradient or any other method that provides a single cell suspension.

Decant the supernatant.

Resuspend the cells in cold 1X MagCellect Plus Buffer.

Decant the supernatant.

Perform a cell count.

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.

Transfer 20 x 10 8 cells (1.0 mL) into a 5-mL polystyrene tube.

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 250 uL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Add 0.85 mL of 1X MagCellect Plus Buffer.

Mix gently.

Add 1.6 mL of cold 1X MagCellect Plus Buffer.

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.

Place the reaction tube in the MagCellect Magnet.

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.

Repeat the magnetic depletion with the new tube containing the recovered cells. The supernatant obtained at the end of this step is the final depleted cell fraction containing the desired enriched CD4+ T 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.

Add 10 uL of MagCellect Mouse CD25+ T Cell Biotinylated Antibody Cocktail per 1 x 10 7 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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