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MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit

Analysis of CD24low/–CD44+ Cells Isolated using the MagCellect CD24– CD44+ Breast Cancer Stem Cell Isolation Kit.
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MAGH111
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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. Add 25 μL of Biotinylated CD24 Antibody.
  4. Incubate for 15 minutes at 2 °C to 8 °C.
  5. Wash samples with cold 1X MagCellect Plus Buffer.
  6. Resuspend the 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 1X MagCellect Plus Buffer.
  12. Place the tube in the MagCellect magnet.
  13. Incubate for 6 minutes at room temperature.
  14. Collect the CD24low/- cells by transferring the supernatant to a new tube.
  15. Place the tube in the magnet and repeat the isolation at least one more time.
  16. Centrifuge the recovered cells at 300 x g for 8 minutes.
  17. Resuspend the pellet in MagCellect Plus Buffer.

Isolation of CD44+ cells by Positive Selection


 

 

 

Recovery of CD24low/- CD44+ cells

 

Analysis of Cell Purity

 
  1. Add 10 μL of Biotinylated CD44 Antibody.
  2. Incubate for 15 minutes at 4 °C.
  3. Add 3 mL of cold 1X MagCellect Plus Buffer.
  4. Centrifuge at 300 x g for 8 minutes.
  5. Resuspend the cell pellet in cold 1X MagCellect Plus Buffer.
  6. Add 50 μL of MagCellect Streptavidin Ferrofluid.
  7. Incubate for 15 minutes at 4 °C.
  8. Add 3 mL of cold 1X MagCellect Plus Buffer.
  9. Centrifuge at 300 x g for 8 minutes.
  10. Resuspend the pellet in cold MagCellect Plus Buffer.
  11. Place the reaction tube in the MagCellect magnet.
  12. Incubate for 6 minutes at room temperature.
  13. Aspirate the supernatant to remove the unwanted cells.
  14. Remove the tube from the magnet.
  15. Add 2 mL of cold 1X MagCellect Plus Buffer.
  16. Repeat 6 minute incubation and removal of supernatant at least one more time.
  17. Remove the tube from the magnet.
  18. Resuspend the cells in 1X MagCellect Plus Buffer or media.
  19. Resuspend the appropriate amount of isolated cells in 100 μL of 1X MagCellect Plus Buffer.
  20. Add 10 μL of Human CD24 and CD44 Detection Antibodies.
  21. Stain and analyze cells using standard flow cytometry procedures.

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Product Datasheets

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