Flow Cytometric DNA Analysis of Human Cancer Cell Lines

  • Peter Mullen
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 88)


Flow cytometry is a rapid technique allowing simultaneous analysis of multiple cellular parameters, including DNA content. The technique relies on a single-cell suspension being passed within a stream of sheath fluid through an optically focused excitation light source, either a laser or an arc lamp. The most common excitation wavelength used in flow cytometers is a 488 nm wavelength light from an argon laser. Lasers provide a single wavelength of coherent light while arc lamps produce a mixture of incoherent wavelengths that must be filtered. When a laser light source is used, the amount of light scattered in the forward direction is detected in the forward angle light scatter (FALS) channel and its intensity is roughly proportional to the size of the cells or particles. Light scattered perpendicular to the path of the laser is detected in the side scatter channel (SSC) and its intensity is more closely related to granularity. If the cells have been stained with fluorochromes they will also emit fluorescence intensities at levels that directly correspond to the density of fluorochrome on or within the cell. The fluorescence signal emitted by any specific fluorochrome is collected through separate channel detectors (photomultipliers) by means of a series of optical filters and mirrors that guide the beam of light. In order to simultaneously measure more than one fluorescence signal from any given cell, multiple channels/detectors are used. A more comprehensive resume can be found elsewhere (1).


Trisodium Citrate Sheath Fluid Photo Multiplier Tube Forward Angle Light Scatter Forward Light Scatter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Shapiro, H. M. Practical Flow Cytometry, 4th Ed., Alan R Liss, ISBN 0-471-41125-6.Google Scholar
  2. 2.
    Barlogie, B., Raber, M. N., Schuman, J., et al. (1983) Flow cytometry in clinical cancer research. Cancer Res. 43, 3982–3997.PubMedGoogle Scholar
  3. 3.
    Auer, G. U., Caspersson, T. O., and Wallgren, A. S. (1980) DNA content and survival in mammary cancer. Anal. Quant. Cytol. Histol. 2, 161–165.Google Scholar
  4. 4.
    Baildam, A. D., Zaloudik, J., Howell, A., et al. (1987) DNA analysis by flow cytometry, response to endocrine treatment and prognosis in advanced carcinoma of the breast. Br. J. Cancer 55, 553–559.PubMedCrossRefGoogle Scholar
  5. 5.
    Kallioniomi, O. P., Hietanen, T., Mattila, J., Lentinen, M., Lauslahti, K., and Koivula, T. (1987) Aneuploid DNA cancer and high S-phase fraction of tumour cells are related to poor prognosis in patients with primary breast cancer. Eur. J. Cancer Clin. Oncol. 23, 277–282.CrossRefGoogle Scholar
  6. 6.
    Pinto, A. E., Andre, S., and Soares, J. (1999) Short term significance of DNA ploidy and cell proliferation in breast carcinoma: a multivariate analysis of prognostic markers in a series of 308 patients. J. Clin. Pathol. 52, 604–611.PubMedCrossRefGoogle Scholar
  7. 7.
    Bracko, M., Us-Krasovec, M., Cufer, T., Lamovec, J., Zidar, A., and Goehde, W. (2001) Prognostic significance of DNA ploidy determined by high resolution flow cytometry in breast carcinoma. Anal. Quant. Cytol. Histol. 23, 56–66.PubMedGoogle Scholar
  8. 8.
    Evans, M. P., and Podratz, K. C. (1996) Endometrial neoplasia: prognostic significance of ploidy status. Clin. Obstet. Gynecol. 39, 696–706.PubMedCrossRefGoogle Scholar
  9. 9.
    Ozalp, S., Yalcin, O. T., Gulbas, Z., Tanir, H. M., and Minsin, T. (2001) Effect of cellular DNA content on the prognosis of epithelial ovarian cancers. Gynecol. Obstet. Invest. 52, 93–97.PubMedCrossRefGoogle Scholar
  10. 10.
    Kimmig, R., Wimberger, P., Hillemanns, P., Kapsner, T., Caspari, C., and Hepp, H. (2002) Multivariate analysis of the prognostic significance of DNA-ploidy and S-phase fraction in ovarian cancer determined by flow cytometry following detection of cytokeratin-labelled tumor cells. Gynecol. Oncol. 84, 21–31.PubMedCrossRefGoogle Scholar
  11. 11.
    Fordham, M. V., Burdge, A. H., Matthews, J., Williams, G., and Cooke, T. (1986) Prostatic carcinoma cell DNA content measured by flow cytometry and its relation to clinical outcome. Br. J. Surg. 73, 400–403.PubMedCrossRefGoogle Scholar
  12. 12.
    Wolley, R. C., Schreiber, K., Koss, L. G., Karas, M., and Sherman, A. (1982) DNA distribution in human colon carcinomas and its relationship to clinical behaviour. J. Natl. Cancer Inst. 69, 15–22.PubMedGoogle Scholar
  13. 13.
    Emdin, S. O., Stenling, R., and Roos, G. (1987) Prognostic value of DNA content in colorectal carcinoma. A flow cytometric study with some methodologic aspects. Cancer 60, 1282–1287.PubMedCrossRefGoogle Scholar
  14. 14.
    Lanza, G., Gafa, R., Santini, A., et al. (1998) Prognostic significance of DNA ploidy in patients with stage II and stage III colon carcinoma: a prospective flow cytometric study. Cancer 82, 49–59.PubMedCrossRefGoogle Scholar
  15. 15.
    Salud, A., Porcel, J. M., Raikundalia, B., Camplejohn, R. S., and Taub, N. A. (1999) Prognostic significance of DNA ploidy, S-phase fraction, and P-glycoprotein expression in colorectal cancer. J. Surg. Oncol. 72, 167–174.PubMedCrossRefGoogle Scholar
  16. 16.
    Deans, G. T., Williamson, K., Heatley, M., et al. (1993) The role of flow cytometry in carcinoma of the colon and rectum. Surg. Gynecol. Obstet. 177, 377–382.PubMedGoogle Scholar
  17. 17.
    Resnik, E., Trujillo, Y. P., and Taxy, J. B. (1997) Long term survival and DNA ploidy in advanced epithelial ovarian cancer. J. Surg. Oncol. 64, 299–303.PubMedCrossRefGoogle Scholar
  18. 18.
    Coley, H. M., Sargent, J. M., Titley, J., and Taylor, C. G. (1999) Lack of prognostic significance of ploidy and S-phase measurements in advanced ovarian cancer. Anticancer Res. 19, 2111–2116.PubMedGoogle Scholar
  19. 19.
    Duigou, F., Herlin, P., Marnay, J., and Michels, J. J. (2000) Variation of flow cytometric DNA measurement in 1,485 primary breast carcinomas according to guidelines for DNA histogram interpretation. Cytometry 15, 35–42.CrossRefGoogle Scholar
  20. 20.
    Emri, S., Akbulut, H., Zorlu, F., et al. (2001) Prognostic significance of flow cytometric DNA analysis in patients with malignant pleural mesothelioma. Lung Cancer 33, 109–114.PubMedCrossRefGoogle Scholar
  21. 21.
    Maillo, A., Diaz, P., Blanco, A., et al. (1999) Proportion of S-phase tumour cells measured by flow cytometry is an independent prognostic factor in meningioma tumors. Cytometry 38, 118–123.PubMedCrossRefGoogle Scholar
  22. 22.
    Koester, S. K., Maenpaa, J. U., Wiebe, V. J., et al. (1994) Flow cytometry: potential utility in monitoring drug effects in breast cancer. Breast Cancer Res. Treat. 32, 57–65.PubMedCrossRefGoogle Scholar
  23. 23.
    Hedley, D. W., Friedlander, M. L., Taylor, I. W., Rugg, C. A., and Musgrove, E. A. (1984) Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31, 1333–1335.Google Scholar
  24. 24.
    Schultz, D. S. and Zarbo, R. J. (1992) Comparison of eight modifications of Hedleys method for flow cytometric DNA ploidy analysis of paraffin-embedded tissue. Am. J. Clin. Pathol. 98, 291–295.PubMedGoogle Scholar
  25. 25.
    Alanen, K. A., Klemi, P. J., Joensuu, H., Kujari, H., and Pekkala, E. (1989) Comparison of fresh, ethanol-preserved, and paraffin-embedded samples in DNA flow cytometry. Cytometry 10, 81–85.PubMedCrossRefGoogle Scholar
  26. 26.
    Levack, P. A., Mullen, P., Anderson, T. J., Miller, W. R., and Forrest, A. P. M. (1987) DNA analysis of breast tumour fine needle aspirates using flow cytometry. Br. J. Cancer 56, 643–646.PubMedCrossRefGoogle Scholar
  27. 27.
    Vindeløv, L. L., Christensen, I. J., Keiding, N., Spang-Thomsen, M., and Nissen, N. I. (1983) Long term storage of samples for flow cytometric DNA analysis. Cytometry 3, 317–322PubMedCrossRefGoogle Scholar
  28. 28.
    Vindeløv, L. L., Christensen, I. J., Nissen, N. I. (1983) A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3, 323–327.PubMedCrossRefGoogle Scholar
  29. 29.
    Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. T. (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J. Nat. Cancer Inst. 51, 1409–1413.PubMedGoogle Scholar
  30. 30.
    Langdon, S. P., Lawrie, S. S., Hay, F. G., et al. (1988) Characterisation and properties of nine human ovarian cancer cell lines. Cancer Res. 48, 6166–6172.PubMedGoogle Scholar
  31. 31.
    Vindeløv, L. L., Christensen, I. J., and Nissen, N. I. (1983) Standardisation of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3, 328–331.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Peter Mullen
    • 1
  1. 1.Cancer Research UK Oncology UnitWestern General HospitalEdinburghUK

Personalised recommendations