Isolate or Deplete Human Epithelial Cells from Whole Blood, Buffy Coat, Bone Marrow, MNC, Tissue Samples

Introduction

Isolate or deplete human epithelial cells directly from whole blood, buffy coat, bone marrow, MNC or tissue samples with Dynabeads® Epithelial Enrich (1, 2). The enriched cells are viable and may be used in cellular applications or lysed for further molecular analysis such as mRNA isolation and RT-PCR. (3,4,5,6,7,8)

For flow-based analysis we recommend Dynabeads® CELLection™ Epithelial Enrich (cat. no. 162.03).

Circulating tumour epithelial cells are extremely rare and may be present at a frequency of only 1-10 in 106 MNC, and an enrichment step is usually essential to obtain a sufficient specificity and sensitivity for tumor cells (5,6,9,10,1 1).

Principle of Isolation

Dynabeads® are mixed with the sample in a tube. The Dynabeads® will bind to the target cells during a short incubation, and then the bead-bound cells can subsequently be separated by a magnet. Dynabeads® are then mixed with the cell sample in a tube.

  • Positive isolation – discard the supernatant and use the bead-bound cells for downstream applications (e.g. cell culture, protein- or gene expression analysis). For further molecular applications, the epithelial cells may be lysed while they are still attached to the beads.
  • Depletion – discard the bead-bound cells and use the remaining, untouched cells for any application.


Description of Materials

Dynabeads® Epithelial Enrich are uniform, superparamagnetic, polystyrene beads (4.5 μm diameter) coated with a mouse IgG1 monoclonal antibody (clone Ber-EP4) specific for two (34 and 39 kDa) glycopolypeptide membrane antigens expressed on most normal and neoplastic human epithelial tissues (12). The reactivity of this antibody  identical to mAb clone HEA125.

Materials Supplied

5 ml Dynabeads® Epithelial Enrich

4 x 108 beads/ml in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3).

Product Capacity

Cat No. Product Volume Sample Numbers Volume of beads used per sample
161.02   
 5 ml40 samples of 5 ml blood125 μl Dynabeads® per 5 ml blood sample
  200 samples of 2 x 107 MNC25 μl Dynabeads® per 2 x 107 MNC


* Sample numbers do not include controls

Additional Materials Required

  • Magnet (e.g. DynaMag™) and mixer allowing both tilting and rotation of test tubes. For magnet and mixer recommendations
  • Washing and isolation buffer: PBS w/ 0.1% BSA and 0.6% sodium citrate (without Ca2+ and Mg2+), pH 7,4.


Important Notes

  • It is critical to keep the cell suspension and buffer cold (2-8°C) during incubation and separation procedures, to prevent phagocytosis of Dynabeads®.
  • Bead-bound cells must be handled gently. Avoid excess pipetting.
  • PBS containing Ca 2+ or Mg 2+ is not recommended.
  • BSA can be replaced by HSA or FCS.  Sodium citrate can be replaced by EDTA.
  • Follow the magnet recommendations to ensure a successful isolation.

Protocols

Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

  1.    Resuspend the Dynabeads® in the vial.

  2.    Transfer the desired volume of Dynabeads® to a tube.

  3.    Add the same volume of buffer (PBS w/ 0.1% BSA and 0.6% sodium citrate), or at least 1 ml, and mix.

  4.    Place the tube in a magnet for 1 minute and discard the supernatant.

  5.    Remove the tube from the magnet and resuspend the Dynabeads® in the original volume (step 2) using the buffer.

Sample Preparation

Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC or tissue digests. MNC (e.g. from bone marrow or blood) Prepare a mononuclear cell suspension. Dilute MNC with buffer to a concentration of 1-2 x 107 cells/ml. Keep on ice.

Whole blood and Buffy Coat

Cool 5 ml of anticoagulated whole blood (EDTA or ACD). Keep on ice. 2.3 Positive Isolation of Tumour Cells MNC

  1. Add 25 μl (1 x 107) washed Dynabeads® Epithelial Enrich to each 1 ml MNC cell sample and leave on a roller for 30 minutes at 2-8°C.

  2. Place the tube in the magnet for 3 minutes at 2-8°C. Invert the magnet with the tube gently after 1 minute, in order to collect beads left in the cap of the tube.

  3. Remove the supernatant carefully by pipetting.

  4. Add 800 μl cold buffer (PBS w/ 0.1% BSA and 0.6% sodium citrate). Remove the tube from the magnet and resuspend the bead-bound cells carefully and transfer the suspension to a new tube.

  5. Place the tube on the magnet for 3 minutes. Remove the supernatant without disturbing the bead pellet.

  6. Add 800 μl of cold buffer to the tube and remove the tube from the magnet. Gently resuspend the bead/cell complexes by pipetting.

  7. Repeat steps 5 and 6 three times, i.e. a total of 5 washes.

  8. Transfer the bead suspension to a new tube. Keep on ice for immediate downstream applications


Whole blood and Buffy Coat

  1. Add 125 μl (5 x 107) washed Dynabeads® Epithelial Enrich to each 5 ml blood sample and leave on roller for 30 minutes at 2-8°C.

  2. Place the tube in the magnet for 5 minutes at 2-8°C. Invert the magnet with the tube after 3 minutes, in order to collect beads left in the cap of the tube.

  3. Remove the blood supernatant carefully by pipetting.

  4. Add 1 ml cold buffer (PBS w/ 0.1% BSA and 0.6% sodium citrate) while the tube is still on the magnet, in order to remove blood left in the tube and on the tube walls. Avoid disturbing the bead pellet.

  5. Discard the buffer.

  6. Add 800 μl cold buffer. Remove the tube from the magnet and resuspend the bead-bound cells carefully.

  7. Transfer the suspension to a new tube.

  8. Place the tube in the magnet for 3 minutes. Remove the supernatant.

  9. Add 800 μl of cold buffer to the tube and remove the tube from the magnet. Gently resuspend the bead/cell complexes by pipetting.

  10. Place the tube in the magnet for 3 minutes.

  11. Repeat steps 9-10, three times, i.e. a total of 5 washes.

  12. Transfer the bead suspension to a new tube. Keep on ice for immediate downstream applications (see Isolate or Deplete Human Epithelial Cells from Whole Blood, Buffy Coat, Bone Marrow, MNC, Tissue Samples).

General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .

References

  1. Hempel D et al. Efficacy of monoclonal antibody (MOAB) HEA125 in enrichment of cancer cells from bone marrow and stem cell collections for quantification of minimal residual disease load and further immunocytochemical characterization of cancer cells. Stem Cell Transplantation, San Diego, April 1996.

  2. Wu H et al. (2005) Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature. 438: 981-987.

  3. Hardingham JE et al. (1995) Detection of circulating tumour cells in colorectal cancer by immunobead- PCR is a sensitive prognostic marker for relapse of disease. Molecular Medicine. 1(7):789-794.

  4. Hardingham JE et al. (1993) Immunobead- PCR: A technique for the detection of circulating tumor cells using immunomagnetic beads and the polymerase chain reaction. Cancer Research. 53:3455-3458.

  5. Cremoux P et al. (2000) Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patient by realtime polymerase chain reaction. Clin. cancer research. 6 (8):3117-3122.

  6. Sakaguchi M et al. (1999) Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial cells in effusions. British Journal of Cancer. 79(3/4):416-422.

  7. Park et al. (2001) Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood. J Cancer Res Clin Oncol. 127:489.

  8. Guo J et al. (2005) Detection of cytokeratin 20 mRNA in the peripheral blood of patients with colorectal cancer by immunomagnetic bead enrichment and real-time reverse transcriptase– polymerase chain reaction. Journal of Gastroenterology and Hepatology. 20:8,1279.

  9. LR Gauthier et al. (2001) Detection of circulating carcinoma cells by telomerase activity. Br. J. Cancer. 84 (5):631-635.

  10. J-C Soria et al. (1999) Molecular detection of telomerase-positive circulating epithelial cells in metastatic breast cancer patients. Clinical Cancer Research. 5:971-975.

  11. Naume B et al. (1997) Immunomagnetic techniques for enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. Journal of Hematotherapy. 6:103-113.

  12. Latza U et al. (1990) Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelia. J. Clin. Pathol. 43: 213-219.


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161.02.indd   Rev 005    5-May-2009

For Research Use Only. Not for use in diagnostic procedures.