Flow Cytometric DNA Analysis of Human Cancer Cell Lines
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).
KeywordsSucrose DMSO Argon Citrate Syringe
- 1.Shapiro, H. M. Practical Flow Cytometry, 4th Ed., Alan R Liss, ISBN 0-471-41125-6.Google Scholar
- 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
- 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
- 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