Mouse Lineage Depletion Protocol

Monoclonal anti-mouse CD3, CD4, CD5, CD8α , CD11b/MAC-1α , B220, Gr-1 (L?-6G) and TER-119 erythroid antigen antibodies can be used to efficiently stain 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 for the enrichment of mouse uncommitted hematopoietic progenitors.

Supplies Required

Materials

• Mice, 6 - 8 weeks old
• 50 mL centrifuge tubes (Falcon Catalog # 352098 or equivalent)
• 15 mL centrifuge tubes (Corning Costar Catalog # 430052 or equivalent)
• 0.2 µm, 500 mL filter units (Nalgene Catalog # 161-0020 or equivalent)
• 0.2 µm syringe filters (PALL Corporation Part # 4187 or equivalent)
• 10 mL syringes (Becton Dickinson Catalog # 309604 or equivalent)
• 70 µm cell strainers (Falcon Catalog # 352350 or equivalent)
• 5 mL polypropylene tubes, 12 x 75 mm with snapcaps (Falcon Catalog # 352063 or equivalent)
• 2 mL polypropylene microtubes (Sarstedt Catalog # 72694.006 or equivalent)
• Scalpel blades
• 21-gauge needles
• 23-gauge needles
• 25-gauge needles
• Serological pipettes
• Pipettors and pipette tips

Reagents

• Monoclonal anti-mouse CD3, CD4, CD5, CD8α , CD11b/MAC-1α , B220, Gr-1 (L?-6G) and TER-119 erythroid antigen antibodies (R&D Systems Catalog # MLDP1, MLDP2, MLDP3, MLDP4, MLDP5, MLDP7, MLDP6 and MLDP8, respectively)
• Phosphate-Buffered Saline (PBS) (Invitrogen Catalog # 10010-023 or equivalent)
• BSA, very low endotoxin (Serologicals Proteins Inc. Catalog # 81-068-3 or equivalent)
• Fetal Bovine Serum (FBS), ES Cell Qualified (Invitrogen Catalog # 16141-061 or equivalent)
• Sterile, deionized water
• Mouse Erythrocyte Lysing Kit (R&D Systems Catalog # WL2000 or equivalent)
• Dynabeads M-450 Sheep anti-rat IgG (Invitrogen Catalog # 110-35)
• Anti-rat IgG-FITC (Caltag Catalog # R40101 or equivalent) or anti-rat IgG-PE (Caltag Catalog # R40104 or equivalent)
• Equipment
• 4° C Centrifuge
• Rotator
• Hemocytometer
• Microscope
• Magnetic particle concentrator (Invitrogen Catalog # 110-35)

Reagent Preparation

Depletion Antibody Cocktail - Mix 12.5 mL of each of the monoclonal anti-mouse CD3, CD4, CD5, CD8α , CD11b/MAC-1α , B220, Gr-1 (L?-6G) and TER-119 erythroid antigen antibodies in a 2 mL polypropylene microtube to make 100 µL of antibody cocktail (sufficient to deplete lineage positive cells from 1 x 10⁸ bone marrow cells). Mix gently. Prepare fresh as needed.

PBS with 2% FBS - Add 10 mL of FBS to 490 mL of PBS to make 500 mL of PBS with 2% FBS. Sterile filter the solution using a 0.2 µm, 500 mL filter unit. Store at 2 - 8°C for up to 1 month.

M-lyse Buffer (optional) - Add 1 mL of 10X M-lyse Buffer (contained in the Mouse Erythrocyte Lysing Kit) to 9 mL of sterile, distilled water. Store at 2 - 8°C for up to one year.

Preparation of Bone Marrow Cells

Use sterile technique if the cells are to be used for in vitro culture or in vivo assays after depletion.

• Harvest the hind leg bones and collect the bone marrow cells of mice. For a detailed protocol, please refer to reference 1.
• Centrifuge the cells for 5 minutes at 400 x g and 4°C. Remove the supernate and resuspend the cells in 10 mL of cold PBS with 2% FBS by gentle pipetting, to generate a single cell suspension. Filter the cell suspension through a 70 µm cell strainer to remove cell clumps.
• After filtration, the cells can be used directly for depletion. Or, if desired, the red blood cells (RBC) can be removed. To remove RBC, resuspend the cells in 5 mL of M-lyse Buffer followed by centrifugation for 5 minutes at 400 x g and 4°C. Remove the supernate and resuspend the cells in cold PBS with 2% FBS. Count the viable cells. After RBC lysis, one mouse typically yields 30 - 60 x 10⁶ cells.

Lineage Depletion of Bone Marrow Cells Followed by negative selection with Dynabeads®

• After the viable cell number is determined, resuspend the cells in cold PBS with 2% FBS to a final density of 1x10⁸ cells/400 µL. Transfer the cells to a 5 mL polypropylene tube. Retain approximately 0.5 x 10⁶ cells to stain with anti-rat IgG-FITC or -PE as a control.
• Add 100 µL of Depletion Antibody Cocktail to each 400 µL of cells. Incubate the cells on ice for 40 minutes. Mix the cells every 10 minutes.
• Wash the cells 3 times with 3 mL of cold PBS with 2% FBS to remove excess antibody by centrifugation at 400 x g for 10 minutes. Retain approximately 0.5 x 10 6 cells to stain with a suitable anti-rat IgG-FITC or -PE conjugate to assess the proportion of lineage positive cells. Compare this sample with the cell preparation at the end of the procedure to determine the depletion efficiency.
• Remove the supernate by pipetting and suspend the cells in 1 mL of cold PBS with 2% FBS.
• Prepare Dynabeads M-450 Sheep anti-rat IgG by calculating the volume of beads needed based on a bead to cell ratio of 2:1. Wash the appropriate volume of beads twice with 3 mL of cold PBS with 2% FBS, capturing the beads with a magnet after each wash. Resuspend the beads in 200 m L PBS with 2% FBS.
• Add 100 mL of washed beads to the cells from step 4. Incubate on ice bath for 5 minutes with constant hand agitation. Add cold PBS with 2% FBS to bring the volume up to 3 mL. Cap the tube and rotate end over end for 25 minutes at 2-8°C to facilitate cell binding.
• Capture the lineage-positive cell-bound beads using a magnet for 2 minutes. Transfer the unbound, lineage-negative cells, using a sterile plastic pipette (do not use a glass pipette), to a 5 mL polypropylene tube. Discard the beads. Centrifuge the lineage-negative cells for 5 minutes at 400 x g and 4°C
• Discard the supernate and resuspend the lineage-negative cells in 1 mL cold PBS with 2% FBS. Add the remaining 100 mL of washed beads and incubate as in step 6.
• Capture the residual lineage-positive cells and harvest the lineage-negative cells as in step 7. Count the viable cells. This procedure removes approximately 98-99% of the lineage-positive cells.
• Label a small aliquot of the lineage-negative cells with an appropriate dilution of anti-rat-IgG-FITC or -PE conjugated to determine the efficiency of lineage-positive cell removal. Compare the efficiency with that obtained from the aliquot of cells stained in the identical manner from step 3. See Figure 1 below for an example of lineage depletion using the depletion antibody cocktail. After depletion the lineage-negative cells contain enriched CD117 (SCF R/c-kit) cells (typically 70-90%) and less than 2% of lineage-positive contamination.

A	B	C
Relative Fluorescence-FITC	Fluorescence Intensity-PE	Fluorescence Intensity-PE
Figure 1: Lineage positive bone marrow cells from C57B1/6 mice were depleted using monoclonal anti-mouse CD3, CD4, CD5, CD8α, CD11b/MAC-1α, B220, Gr-1 (L?-6G) and TER-119 erythroid antigen antibodies and stained with goat anti-rat IgG PE-conjugated. Flow cytometric analysis was performed A) Before (dotted histogram) and after (solid histogram) the depletion antibody cocktail was added; B) Before (dotted histogram) and after (solid histogram) magnetic depletion using Dynabeads; and C) Expression of CD117 (SCF R/c-kit) (R&D Systems Catalog # MAB1356; dotted histogram) in bone marrow-progenitor cells after enrichment using the mouse lineage panel and bead depletion.

Reference

1. Current Protocols in Immunology, Isolation of Murine Macrophages, copyright 1994, Coligan, J.E. et al. eds., John Wiley & Sons, Inc., Volume 3, Supplement 11, 14.1.4.