MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit

Catalog # Availability Size / Price Qty
MAGH111
Product Details
Procedure
Citations (1)
FAQs
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MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit Summary

Kit Summary

For the isolation of breast cancer stem cells.

Key Benefits

  • Isolation by positive and negative selection improves purity of recovered cells
  • Isolation based on two markers increases the purity of recovered cells
  • Includes antibodies for analysis of the final cell population purity

Why use Both Positive (CD44) and Negative (CD24) Selection to Isolate Breast Cancer Stem Cells?

Cancer stem cells are a rare population of self-renewing cells found in tumors and in cancer cell lines. Methods that use positive or negative selection to enrich for cancer stem cells can lead to poor recovery and low cell purity as well as the loss of valuable tissue samples and resources.  See Details

Methods that use both positive and negative selection to isolate cancer stem cells can yield cells with a higher purity.

R&D Systems MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit (Catalog # MAGH111) enriches for a small population of human breast cancer cells, which can form new tumors in mice. This kit uses a two-step procedure that combines negative and positive selection techniques. Briefly, CD24low/– cells are initially enriched by negative selection whereby unwanted CD24+ cells are tagged and magnetically removed. CD44+ cells are subsequently isolated from the CD24low/– population by positive selection using a biotinylated human CD44 antibody, streptavidin-conjugated magnetic beads, and a MagCellect magnet (Catalog # MAG997, or equivalent). The efficiency of enrichment can be assessed by staining the recovered cells with fluorochrome-conjugated anti-human CD24 and CD44 detection antibodies which are also provided in the kit.

The MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit:

  • Isolates CD24low/- CD44+ cells which have been shown to form tumors in mice.
  • Uses positive and negative selection to increase the purity of isolated cells.
  • Includes fluorochrome-conjugated CD24 and CD44 antibodies for analysis of recovered cells.

Components

  • APC-conjugated Mouse Anti-Human CD24 Detection Antibody - 0.2 mL (20 tests)
  • Mouse Anti-Human CD24 Biotinylated Antigen Affinity-purified Antibody – 0.5 mL (20 tests)
  • Mouse Anti-Human CD44 Biotinylated Antigen Affinity-purified Antibody – 0.2 mL (20 tests)
  • PE-conjugated Mouse Anti-Human CD44 Detection Antibody - 0.2 mL (20 tests)
  • MagCellect Streptavidin Ferrofluid – 2 mL
  • 10X MagCellect Plus Buffer – 50 mL

Storage

Reagents are stable for 12 months from the date of receipt when stored in the dark at 2 °C to 8 °C. DO NOT FREEZE.

Precaution

  • The PE- and APC-conjugated detection antibodies provided in this kit contain sodium azide which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.

 

Data Examples


View Larger Image

Analysis of CD24low/–CD44+ Cells Isolated using the MagCellect CD24 CD44+ Breast Cancer Stem Cell Isolation Kit. A small population of CD24low/–CD44+ cells was isolated from MCF-7 human breast adenocarcinoma cells using the MagCellect CD24 CD44+ Breast Cancer Stem Cell Isolation Kit (Catalog # MAGH111). CD24low/–CD44+ cells (upper left quadrant), before (A) and after (B) enrichment, were detected by double-staining with APC-conjugated Mouse Anti-Human CD24 and PE-conjugated Mouse Anti-Human CD44 Detection Antibodies (both provided in the kit). A histogram profiling the enrichment of CD24low/– cells (filled histogram) from the original cell population (open histogram) is shown (C).

Product Datasheets

Preparation and Storage

Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, CD24low/- CD44+ Breast Cancer Stem Cells can be isolated using the following procedure:

  • Isolate CD24low/- cells by negative selection using the biotinylated CD24 antibody
  • Isolate CD44+ cells from the CD24low/- population of cells using the biotinylated CD44 antibody
  • The purity of the recovered cells can be analyzed using the antibodies included in the kit
 

Reagents Provided

Reagents supplied in the MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit (Catalog # MAGH111):

  • APC-conjugated Mouse Anti-Human CD24 Detection Antibody - 0.2 mL (20 tests)
  • Mouse Anti-Human CD24 Biotinylated Antigen Affinity-purified Antibody – 0.5 mL (20 tests)
  • Mouse Anti-Human CD44 Biotinylated Antigen Affinity-purified Antibody – 0.2 mL (20 tests)
  • PE-conjugated Mouse Anti-Human CD44 Detection Antibody - 0.2 mL (20 tests)
  • MagCellect Streptavidin Ferrofluid – 2 mL
  • 10X MagCellect Plus Buffer – 50 mL

Other Supplies Required

Reagents

  • Deionized or distilled water

Materials

  • MagCellect Magnet (Catalog # MAG997)
  • 12 x 75 mm (5 mL) or 17 x 100 mm (15 mL) polystyrene round bottom tubes
  • Sterile Pasteur pipettes or transfer pipettes

Equipment

  • Benchtop centrifuge
  • 2 °C to 8 °C refrigerator
 

Note: While optimized for R&D Systems reagents and supplies, MagCellect kits have been tested in combination with EasySep™ (StemCell Technologies), iMag™ (Becton Dickinson), and Streptavidin Microbeads™ (Miltenyi Biotec) magnetic beads and magnets. When using other supplier’s magnetic selection systems, the protocol may need to be adapted according to the supplier’s directions for optimal performance.

Protocol for Isolation of Breast Cancer Stem Cells Using the MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit (Catalog # MAGH111):

Removal of CD24+ Cells by Negative Selection


  1. Resuspend cells in cold 1X MagCellect Plus Buffer.
  2. Place 5 x 106 cells (0.5 mL) into a 5 mL round bottom tube
  3.  

  4. Add 25 μL of Biotinylated CD24 Antibody.
  5. Incubate for 15 minutes at 2 °C to 8 °C.
  6.  

  7. Wash samples with cold 1X MagCellect Plus Buffer.
  8. Resuspend the pellet in cold 1X MagCellect Plus Buffer.
  9. Add 50 μL of MagCellect Streptavidin Ferrofluid.
  10. Incubate for 15 minutes at 4 °C.
  11. Add 3 mL of cold 1X MagCellect Plus Buffer.
  12. Centrifuge at 300 x g for 8 minutes.
  13. Resuspend the pellet in cold 1X MagCellect Plus Buffer.
  14.  

  15. Place the tube in the MagCellect magnet.
  16. Incubate for 6 minutes at room temperature.
  17. Collect the CD24low/- cells by transferring the supernatant to a new tube.
  18. Place the tube in the magnet and repeat the isolation at least one more time.
  19.  

  20. Centrifuge the recovered cells at 300 x g for 8 minutes.
  21. Resuspend the pellet in MagCellect Plus Buffer.
  22.  

Isolation of CD44+ cells by Positive Selection


  1. Add 10 μL of Biotinylated CD44 Antibody.
  2. Incubate for 15 minutes at 4 °C.
  3.  

  4. Add 3 mL of cold 1X MagCellect Plus Buffer.
  5. Centrifuge at 300 x g for 8 minutes.
  6. Resuspend the cell pellet in cold 1X MagCellect Plus Buffer.
  7. Add 50 μL of MagCellect Streptavidin Ferrofluid.
  8. Incubate for 15 minutes at 4 °C.
  9. Add 3 mL of cold 1X MagCellect Plus Buffer.
  10. Centrifuge at 300 x g for 8 minutes.
  11. Resuspend the pellet in cold MagCellect Plus Buffer.
  12.  

  13. Place the reaction tube in the MagCellect magnet.
  14. Incubate for 6 minutes at room temperature.
  15. Aspirate the supernatant to remove the unwanted cells.
  16. Remove the tube from the magnet.
  17. Add 2 mL of cold 1X MagCellect Plus Buffer.
  18. Repeat 6 minute incubation and removal of supernatant at least one more time.
  19.  

    Recovery of CD24low/- CD44+ cells

  20. Remove the tube from the magnet.
  21. Resuspend the cells in 1X MagCellect Plus Buffer or media.
  22.  

    Analysis of Cell Purity

  23. Resuspend the appropriate amount of isolated cells in 100 μL of 1X MagCellect Plus Buffer.
  24. Add 10 μL of Human CD24 and CD44 Detection Antibodies.
  25. Stain and analyze cells using standard flow cytometry procedures.
  26.  

Citation for MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. The effect of cycling hypoxia on MCF-7 cancer stem cells and the impact of their microenvironment on angiogenesis using human umbilical vein endothelial cells (HUVECs) as a model
    Authors: FM Alhawarat, HM Hammad, MS Hijjawi, AS Sharab, DA Abuarqoub, MA Al Shhab, MA Zihlif
    PeerJ, 2019;7(0):e5990.  2019

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