MagCellect CD24- CD44+ Breast Cancer Stem Cell Isolation Kit

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, CD24^low/- CD44⁺ Breast Cancer Stem Cells can be isolated using the following procedure:

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

View Graphic Procedure

 

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 – 3 mL
• 10X MagCellect Plus Buffer – 50 mL

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

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 CD24^low/- 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 CD24^low/- 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.

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.

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