To isolate hematopoietic non-lineage committed cells (lineage-negative) from mouse bone marrow.
Less than 5% residual lineage-positive cells remain after depletion
Isolated HSCs are untouched by beads or antibodies
Negative selection reduces sources of experimental variation
Why Isolate Lineage Negative Hematopoietic Cells by Negative Selection?
Both positive and negative selection methods can be used to generate cell suspensions with high purity. However, cells enriched by positive selection can generate cells that are labeled with antibodies and/or beads
These modifications can introduce experimental variability and can invalidate the use of certain antibodies in downstream applications such as flow cytometry. To efficiently obtain untouched cells of high purity by negative selection, R&D Systems offers the MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit. The MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit uses a magnet and a cocktail of antibodies to remove unwanted, lineage positive cells from a cell suspension. Non-lineage committed mouse hematopoietic cells isolated with the kit are untouched by antibodies or magnetic particles and can be used for any downstream application.
Magnetic depletion of lineage-committed mouse hematopoietic cells:
Enriches SCF R/c-kit/CD117+ cells to a purity of 40-70%.
Results in less than 5% residual lineage-positive cells remaining after depletion.
Targets these lineage-committed cells (lineage positive) for depletion:
Does not introduce added sources of experimental variability associated with positive selection.
The MagCellect Mouse Hematopoietic Cell Lineage Depletion kit contains the following reagents for isolation of uncommitted hematopoietic cells from mouse bone marrow:
A cocktail of biotinylated Rat Anti-Mouse Monoclonal Antibodies against CD5, TER-119, CD11b, CD45R/B220, and Ly-6G/Gr-1
*This kit requires the MagCellect Magnet; sold separately (Catalog # MAG997 or equivalent).
Stability and Storage
Store all reagents at 2 °C to 8 °C. DO NOT FREEZE.
The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage depletion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.
MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit Scientific Data Examples
Enrichment of SCF R/c-kit in Bone Marrow Lineage-negative Cells.
SCF R/c-kit staining of bone marrow lineage-negative cells isolated from BALB/c mice using the MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit (Catalog # MAGM209). Histograms reflect the reactivity of all viable cells with a PE-conjugated Rat Anti-Mouse SCF R/c-kit Monoclonal Antibody (Catalog # FAB1356P; filled histogram) or a PE-conjugated Rat IgG2A Isotype Control (Catalog # IC006P; open histogram) before (A) or (B) after lineage depletion.
Depletion of Lineage-committed Hematopoietic Cells.
Lineage marker reactivity on BALB/c bone marrow (BM) cells processed with the MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit (Catalog # MAGM209). The histograms show the reactivity of BM cells labeled with the cocktail of biotinylated antibodies (Rat Anti-Mouse CD5, CD11b, B220/CD45R, Gr-1/Ly-6G, and TER-119) included in the kit both before (green histogram) and after (purple histogram) magnetic depletion. Lineage marker reactivity was detected using Streptavidin-PE.
Preparation & Storage
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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
Resuspend the cells in 1 mL of cold 1X MagCellect Buffer.
Add 150 µL of MagCellect Streptavidin Ferrofluid to the cell suspension.
Incubate at 2 °C to 8 °C for 15 minutes.
Add 0.85 mL of 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.
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.
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.
Which Brands are Currently Available on bio-techne.com? R&D Systems, Tocris Bioscience and ProteinSimple branded products are available to purchase through bio‑techne.com. ProteinSimple branded instruments and Simple Plex Assays are available to quote. ProteinSimple branded instrument consumables and ACD branded products will be available on bio-techne.com in the near future. Novus Biologicals branded products are not currently available on bio‑techne.com and can be found at novusbio.com.
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