Mouse Hematopoietic Lineage Depletion

Antibodies including anti-mouse CD5, CD11b, CD45R (B220), Ly-6G (Gr-1), and TER-119 can be used to efficiently tag bone marrow-derived cells committed to major hematopoietic lineages, including T lymphocytes, B lymphocytes, monocytes/macrophages, granulocytes, and erythrocytes. These antibodies can be used in conjunction with magnetic particle separation systems or flow cytometric cell sorting to deplete lineage-committed cells and enrich for uncommitted hematopoietic progenitors. This procedure describes the depletion of lineage committed cells using the MagCellect™ Mouse Hematopoietic Lineage Depletion Kit (Catalog # MAGM209). The resulting cell population is enriched for CD117⁺ cells (from 40 - 70% depending on mouse strain) with less than 5% residual lineage positive cells.

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• MagCellect Mouse Hematopoietic Lineage Depletion Kit (R&D Systems, Catalog # MAGM209)
• PBS
• Fetal bovine serum (FBS)
• Sterile deionized water

Materials

• 50 mL centrifuge tubes
• 15 mL centrifuge tubes
• Round-bottom Polypropylene tubes: 12 x 75 mm (5 mL) or 17 x 100 mm (15 mL)
• 0.2 µm, 500 mL filter units
• 70 µm cell strainers
• Sterile Pasteur pipettes
• Pipettes and pipette tips

Equipment

• Centrifuge
• Hemocytometer
• Microscope
• MagCellect Magnet (R&D Systems, Catalog # MAG997)

Reagent & Media Preparation

• PBS with 2% FBS - Add 10 mL of FBS to 490 mL of PBS. Sterile filter the solution using a 0.2 µm, 500 mL filter unit. Store at 2 - 8° C for up to 1 month.
• Preparation of MagCellect Buffer - Prepare 25 mL of 1x MagCellect Buffer for each 1.0 x 10⁸ cells to be processed by mixing 2.5 mL of MagCellect 10x Buffer with 22.5 mL sterile deionized or distilled water. The 1x MagCellect Buffer should be kept on ice (or refrigerated) and used within 24 hours.
• Preparation of Bone Marrow Cells - Prepare a suspension of mononuclear cells from mouse bone marrow using traditional methods. Both femurs and tibiae from one mouse typically yield 2.0 - 6.0 x 10⁷ hematopoietic cells, 0.3 - 1.0 x 10⁶ of which are lineage negative. A detailed protocol can be found in Current Protocols in Immunology, Isolation of Murine Macrophages (1994), Coligan, J.E. et al. eds. John Wiley & Sons, Inc., Volume 3, Supplement 11, 14.1.4. To remove cell clumps and debris after harvesting the bone marrow cells, pass the cell suspension through a 70 µm nylon cell strainer.
  Wash the cells in 50 mL centrifuge tubes with cold PBS with 2% FBS by centrifuging at 300 x g for 8 minutes at 2 - 8° C. Remove the supernatant completely and resuspend the cells in 2 mL cold 1X MagCellect Buffer. Count the cells and bring the cell suspension to 2 x 10⁷ cells/mL in 1x MagCellect Buffer. Lysing of red blood cells is not required.
  Note: Reaction incubations must be carried out at 2 - 8° C in a refrigerator and not in ice baths to avoid excessively low temperatures that can slow optimized kinetic reactions.
  Note: If sterile cells are required following lineage depletion, 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.
  Note: 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 labeling, thus lowering cell purity and yield.

Procedure (Figure 1)

1. Lineage Depletion of Bone Marrow Cells using the MagCellect Mouse Hematopoietic Lineage Depletion Kit
   1. Place 1 x 10⁸ cells (5.0 mL volume) into a sterile 15 mL centrifuge tube. Add 50 µL of MagCellect Blocking Reagent 1. Mix gently and incubate at 2 - 8° C in a refrigerator for 15 minutes.
      Note: See below for processing different cell numbers.
   2. Add 100 µL of Lineage Depletion Biotinylated Antibody Cocktail. Mix gently and incubate at 2 - 8° C in a refrigerator for 15 minutes.
   3. Wash away excess antibody by filling the tube to the 15 mL mark with cold 1x MagCellect Buffer and centrifuge at 300 x g for 8 minutes.
   4. Remove the supernatant completely and resuspend the cell pellet with 1 mL of cold 1x MagCellect Buffer by gently pipetting up and down.
   5. Transfer the cells to a clean 5 mL tube (12 x 75 mm). Retain 0.5 x 10⁶ cells to assess the proportion of lineage positive cells by staining with a suitable streptavidin-fluorochrome conjugate.
   6. Add 150 µL of MagCellect Streptavidin Ferrofluid. Mix gently and incubate at 2 - 8° C in a refrigerator for 15 minutes.
   7. At the end of the incubation period bring the volume of the reaction in the tube to 2 mL by adding 0.85 mL of 1x MagCellect Buffer. Mix gently to ensure that all reactants in the tube are in suspension.
   8. Place the reaction tube in the MagCellect Magnet that has been positioned horizontally to accommodate 5 mL tubes, and incubate for 6 minutes at room temperature. Magnetically tagged cells will migrate toward the magnet (these are the unwanted cells), leaving the desired cells suspended in the supernatant.
   9. Recovery of desired cells is achieved as follows: while the tube is in the magnet use a sterile Pasteur pipette or transfer pipette to carefully aspirate all of the reaction supernatant. Place the supernatant in a new 5 mL tube and discard the tube containing the magnetically trapped cells.
   10. To ensure that all of the magnetic nanoparticles have been removed, repeat the magnetic depletion (step # 8 and # 9) 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 lineage negative cells. The cells are now ready for counting, staining or other downstream applications.
       1. When processing different numbers of cells, observe the following guidelines:
          Add 5 µL of Blocking Reagent 1, 10 µL of Antibody Cocktail, and 15 µL of Streptavidin Ferrofluid per each 1 x 10⁷ cells processed.
       2. 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.
       3. When processing greater than 2 x 10⁸ cells:
          Use 17 x 100 mm (15 mL) tubes with the MagCellect Magnet positioned vertically to accommodate up to two 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 magnetic incubation time described in step #8 to 8 minutes. Reaction volume adjustments must be made using 1x MagCellect Buffer just prior to the magnetic separation step.