Flow Cytometers

Abstract

Flow cytometers are analytical instruments that offer an advanced technology for rapidly analysing and separating cells and subcellular components based on their physical, biochemical and functional properties. The technology has broad application in the life sciences, in biomedical research and in the clinic, with the range of applications expanding as cytochemical and instrumental capabilities continue to advance.

Keywords: cytometry; automated cell analysis; cell sorting; analytical cytology; fluorescence; light scatter

Figure 1.

Conceptual diagram of a flow cytometer illustrating the flow chamber; the cell sample and sheath fluid inputs; the laser illumination; the multicolour fluorescence/light scatter (orthogonal) and forward light scatter detectors; the piezoelectric transducer with electrical drive signal input; and the droplet deflection plates and vessels for collecting charged droplets containing separated cells.

Figure 2.

Frequency distribution histograms recorded on ethanol‐fixed CHO cells labelled with Hoechst 33342 (for DNA content) and Pyronin Y (for RNA content) and analysed by dual‐laser flow cytometry using the UV and 530 nm wavelength lines from an argon and a krypton laser, respectively. (a) The UV‐excited Hoechst blue fluorescence (420–500 nm wavelength) intensity histogram. (b) The 530 nm‐excited Pyronin Y yellow fluorescence (560–590 nm wavelength) intensity histogram. (c) The corresponding DNA–RNA content bivariate contour diagram. In (a), peak 1 represents the 2C diploid DNA content of cells in G1 phase of the cell cycle prior to DNA replication, and peak 2 is 4C DNA content of cells in the G2 + M phase following DNA replication. Cells located between the two peaks are in S phase (synthesizing or replicating their DNA).

Figure 3.

Frequency distribution histograms of light scatter intensity measured in the forward direction at 0.5–2.0° relative to the laser excitation beam axis (a) and orthogonally (90°) to the laser beam–cell stream intersection (b). The corresponding bivariate contour diagram of normal unstained leucocytes from a centrifuged human blood buffy coat with the erythrocytes lysed using saponin is shown in (c). The light scatter measurements were made using the 488 nm wavelength line from an argon laser. Regions 1, 2, 3 and 4 are lymphocytes, monocytes, granulocytes (primarily neutrophils) and lysed erythrocytes, respectively.

Figure 4.

Log fluorescence intensity bivariate contour diagram recorded on normal human T lymphocytes labelled with fluorescein isothiocyanate (FITC)‐antiLeu 2a and tetramethylrhodamine isothiocyanate (TmRITC)‐antiLeu 4 monoclonal antibodies and analysed by dual‐laser flow cytometry using the 488 nm and 530 nm wavelength lines from an argon and a krypton laser, respectively. The FITC green (suppressor/cytotoxic T cells) and TmRITC red (total T cells) fluorescence emissions were measured between 520–560 and >580 nm wavelengths, respectively. The Leu 2a and Leu 4 monoclonal antibody designations have subsequently been replaced by CD8 and CD3, respectively. The horizontal and vertical axes (fluorescence intensities) are proportional to log base 10 units (decades).

Figure 5.

(a) Fluorescence intensity frequency distribution histogram recorded on propidium iodide‐stained chromosomes obtained from cultured CCHE cells (Chinese hamster embryo) by mitotic block. The chromosomes were excited using the 488 nm wavelength line from an argon laser and the fluorescence was measured above 515 nm wavelength. (b) Bivariate dot plot diagram of Hoechst 33258 (blue fluorescence) and Chromomycin A3 (yellow fluorescence) stained chromosomes obtained from cultured human lymphoblast GM 2184 cells by mitotic block. The chromosomes were analysed using the UV and 457 nm wavelengths from two argon lasers for exciting Hoechst and Chromomycin, respectively, and the corresponding fluorescence emissions were measuring from 420–500 nm wavelength (blue) and above 515 nm wavelength (yellow). The channel numbers of the horizontal axis of (a) are proportional to red fluorescence intensity.

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Further Reading

Cytometry: The Journal of the International Society for Analytical Cytology (1980–present). New York: Wiley‐Liss.

Givan A (1992) Flow Cytometry First Principles. New York: Wiley‐Liss.

Keller RA, Ambrose WP, Goodwin PM, Jett JH, Martin JC and Wu M (1996) Single molecule fluorescence analysis in solution. Applied Spectroscopy 50: 12A–32A.

Melamed R, Lindmo T and Mendelsohn M (1990) Flow Cytometry and Sorting, 2nd edn. New York: Wiley‐Liss.

Ormerod MG and Poot M (2000) Flow Cytometry: A Practical Approach, 3rd edn. Oxford: Oxford University Press.

Shapiro H (1995) Practical Flow Cytometry, 3rd edn. New York: Wiley‐Liss.

Steinkamp JA (1984) Flow cytometry. Review of Scientific Instruments 55: 1375–1400.

Watson J (1991) Introduction to Flow Cytometry. Cambridge: Cambridge University Press.

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How to Cite close
Steinkamp, John A(Aug 2001) Flow Cytometers. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002971]