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From CellBiology

Flow Cytometry

Introduction

Flow cytometry is a technique for quantifying cellular characteristics as cells in a suspension flow through a sensing point. The advantage of this technique is that each cell in the suspension can be analysed individually, instead of analysing a whole mass of cells. Applications for this method include the analysis of proteins and chromosomes in a cell, and also the study of cell function.

History

  • Flow cytometry developed from microscopy. Thus Leeuwenhoek is often cited in regards to the history of flow cytometry.
  • F.T. Gucker (1947) built the first apparatus for detecting bacteria in a laminar sheath stream of air.
  • L. Kamentsky (IBM Labs), and M. Fulwyler (Los Alamos Nat. Lab.) experimented with fluidic switching and electrostatic cell sorters respectively. Both described cell sorters in 1965.
  • M. Fulwyler utilized Pulse Height Analyzers to accumulate distributions from a Coulter counter. This feature allowed him to apply statistical analysis to samples analyzed by flow.
  • In 1972 L. Herzenberg (Stanford Univ.), developed a cell sorter that separated cells stained with fluorescent antibodies. The Herzenberg group coined the term Fluorescence Activated Cell Sorter (FACS).

Principles of Flow Cytometry

How the machine functions

A schematic of fluorescence detection by a flow cytometer and a schematic of cell sorting by a flow cytometer.

The primary systems that a flow cytometer functions on are fluidics, optics, and electronics. Cells need to be directed into a flow chamber, where the cells are individually analysed one by one as they pass by one or several laser beams. As the light beams hits the cells, it is scattered by the surface of the cell. In addition, fluorescent probes are also used to tag specific cell properties; the laser beam's intensity needs to be able to excite these fluorescent probes accordingly. These scattered beams and fluorescent light generated by the passing cells are collected by photodetectors and converted into electronic signals. The computer is then able to plot these in the relevant dimensions that are being measured.

For a more detailed explanation, see this link: A video-tutorial of the basic principles of Flow Cytometry.

Sample Preparation

  • For the cells to flow through the flow chamber effectively, the sample needs to be produced in a way where all the desired cells are separated, suspended, and stained accordingly (Carter and Meyer, 1994).
  • Body fluids (e.g. blood) generally already are a suspension of individual cells, so the preparation can be more simple.
  • Solid tissue preparations are more complex; relevant techniques used to separate the cells range from:
    • chopping and sieving organ/sample
    • put sample through a density gradient centrifuge
    • enzymatic digestion
    • detergents that remove cell membranes/cytoplasm.

Parameters measured

  • Structural parameters
    • Cell size
    • Cell surface granularity
    • DNA and RNA content
  • Functional parameters
    • DNA synthesis
    • DNA degradation (apoptosis)
    • Cytoplasmic calcium content
    • Gene expression
    • Surface and intracellular receptors

Markers

A cell's function and structure can be characterised by the proteins that are on the cell surface at any particular time. These surface proteins are called antigens, and the proteins that bind on them are called antibodies. These antibodies can be produced by manufacturers for the purpose of flow cytometry, in order to mark specific surface proteins on cells.

Fluorescence

CD4 and CD8 T cells analyzed by flow cytometry.

The technique of fluorescent marking is an important application in flow cytometry (Ormerod, 1994). Fluorescence is the phenomenon which occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength. The emitted light is compared to the exciting light to give data, which is analyzed and interpreted to show certain characteristics of the molecule, and hence structure.

There are two main ways in which fluorescent probes are used in flow cytometry:

  • to label other probes, such as antigens, which are then used to mark cell surfaces;
  • to reflect certain properties of the cell by the way it is distributed.

Examples of fluorophores (fluorescently labelled antibodies):

  • protein labelling - fluorescein isothiocyanate; Texas Red; Peridinin-chlorophyll conjugate
  • nucleic acid labelling - Propidium; ethidium; mithramycin
  • RNA labelling - Pyronin Y; Thiazole Orange; thioflavin T.
  • membrane potential - DiSC3[5]; Rhodamine 123
  • lipid labelling - Acyl aminofluorescein; octadecyl rhodamine B
  • others - Fura-2; indo-1; dichlorofluorescein

More information on Fluorophores.org - The database of fluorescent dyes and applications

Detection Devices

There are two types of devices used in flow cytometers to detect scatter and fluorescence:

  • PIN diodes - Used to detect forward scatter and measure light absorption, these detectors have relatively low sensitivity but wide spectral characteristics and fast response.
  • Photmultiplier tubes (PMTs) - This detector is sensitive to detect weak fluorescence, but has a more restricted spectral response, depending on its photocathode.

Data Analysis

A histogram of flow cytometry data.
A Dot plot of flow cytometry data.

Data generated in flow cytometry can be displayed in various different ways, depending on the dimensions that need to be considered.

1-Dimensional: Frequency histogram

  • This type of plot shows counts against intensity of fluorescence.

2-Dimensional: Cytogram

  • A cytogram, also called a dot plot, measures two parameters against each other, so a comparison can be made. E.g. Forward scatter against side scatter. "Gating" methods can be used to single-out subpopulations to be studied.

Other useful ways of plotting

  • Contour plot - similar to a cytogram, but the points of similar event frequency are joined together, forming contours, which give a somewhat 3-dimensional aspect to the plot.
  • Isometric plot - goes one step further than the contour plot to present the data in a actual 3-D form, allowing rotation and viewing from different tilt angles.
  • Diamond view

Link: A video-tutorial of flow-cytometric data analysis.

Current Applications

Flow cytometry is applied widely in these areas of research:

  • Immunofluorescence
  • Cell cycle kinetics
  • Genetics
  • Molecular Biology
  • Microbiology
  • Bioterrorism

Study of cell division

The advantage of using flow cytometry to study cell division is that the technique allow the cells to be studied with measurement of time parameters, thus providing information about the rates of cell cycle progression (Wilson, 1994). A common fluorescent label for the study of the cell cycle and cell division is bromodeoxyuridine (BrdUrd or BrdU). A concentration of BrdUrd can be added to a cell culture, where it will label the DNA in those cells.

Examples of studies on cell division and cell kinetics, which have been conducted using flow cytometry:

  • Ulrich, H. and Tárnok, A. 2005, 'Quantification of cell-cycle distribution and mitotic index in Hydra by flow cytometry', Cell Proliferation, vol. 38, no. 2, pp. 63-75. PMID: 15842251
  • Dolbeare, F., Selden, J.R. 1994, 'Immunochemical quantitation of bromodeoxyuridine: application to cell-cycle kinetics', 41:297-316. PMID: 7861968
  • Keng, P.C. 1986, 'Use of flow cytometry in the measurement of cell mitotic cycle', International Journal of Cell Cloning, 4(5):295-311. PMID: 2430028
  • Gratzner, H.G. 1982, 'Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.', Science, 218(4571):474-5. PMID: 7123245

Additional Links

References

  1. Carter, N. P. and Meyer, E. W. 1994, 'Introduction to the principles of flow cytometry', in M. G. Ormerod (ed), Flow Cytometry: A Practical Approach, IRL Press, Oxford, pp. 1-25.
  2. Ormerod, M.G. 1994, 'An introduction to fluorescence technology', in M. G. Ormerod (ed), Flow Cytometry: A Practical Approach, IRL Press, Oxford, pp. 27-43.
  3. Nolla, H., 'Basic Principles in Flow Cytometry', A Powerpoint presentation accessed on the Flow Cytometry Facility's website, University of California, Berkeley Campus.[1]
  4. Simmer, M. 2003, "Flow Cytometry: A Technology to count and sort cells". The Science Creative Quarterly, Issue 3. Accessed on http://www.scq.ubc.ca
  5. Wilson, G.D. 1994, 'Analysis of DNA - measurement of cell kinetics by the bromodeoxyuridine/anti-bromodeoxyuridine method', in M. G. Ormerod (ed), Flow Cytometry: A Practical Approach, IRL Press, Oxford, pp. 137-156.



Lecture Feedback

Lecture 8 - Adhesion

What do the different "CAM" acronyms stand for?

  • I-CAM Intercellular Cell Adhesion Molecule
  • L-CAM Liver Cell Adhesion Molecule
  • N-CAM Neural Cell Adhesion Molecule
  • Ng-CAM Neural Glial Cell Adhesion Molecule

Lecture 7 - Mitochondria

What types of cellular processes require lots of energy from the mitochondria?

  • cell movement (eg. flagella)
  • transporting substances over the cell membrane up concentration gradient
  • synthesis of proteins, nucleic acids, and polysaccharides
  • muscle contraction
  • reverse osmosis

Lecture 5 - Exocytosis

What concept about exocytosis did you find difficult to understand?

It was quite easy to understand overall; I just found some of the EM images difficult to label and differentiate different parts of the cell.

Lab 2

Confocal Microscope located at the Histology & Microscopy Unit, School of Medical Sciences, UNSW Sydney. http://medicalsciences.med.unsw.edu.au/SOMSWeb.nsf/page/HMU%20Microscopy%20Services

Electron Microscope located at the Basement of the Chemical Sciences Building, UNSW Sydney. http://srv.emunit.unsw.edu.au/

Lecture 4 - Nucleus

What did you find interesting and did not know about the nucleus?

I found the part on chromosomes interesting. I was reminded that they do not always appear in the structure of a "clothe-peg", but instead, they unfold. I also did not know that different chromosomes occupy different territories within the nucleus. It's amazing how the 24 pairs of chromosomes manage not to tangle up when they are unfolded.

Lecture 10

What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

It is called the stratum spinosum, where desmosomes are found.


Lecture 14 - Confocal Microscopy

What are the 2 main forms of generating confocal microscopy?

Lecture 15 - Cell Cycle

What does "S" stand for in the S phase?

"S" is for synthesis in a cell cycle.