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CHAPTER 4
Molecular and Cellular Probes
INTRODUCTION
T
he concept of cellular identification first arose in the time of Ehrlich and Metchnikoff,
who pioneered diagnostic process in infectious disease (as discussed in Weissman,
1992). Over a century later, histological diagnosis of disease by pathology is the gold
standard to which all other methods are compared. The development of absorptive dyes
to bring out the special features of different cells is the accepted tool of pathologists for
this purpose.
It was only natural to use the light microscope to distinguish between healthy and
unhealthy cells. In the early 1940s antibodies were introduced as a means for more precise
identification (Coons et al., 1941; Moller, 1961). Cytochemical reagents (initially lightabsorbing dyes) were attached to antibodies; when the antibodies bound to cells, a color
reaction was produced at the site of antibody deposition. Later other probes, such as lectins
to surface carbohydrates, were developed as alternatives to antibody probes.
In the early 1960s, fluorescent dyes began to be substituted for light-absorbing dyes,
although primarily only for research specimens. Even today, immunofluorescence is
hardly used by anatomical pathologists. With the association of computers to flow
cytometers and improvements in immunofluorescence detection and instrumentation,
however, flow cytometry using fluorochrome-conjugated antibodies has emerged as a
major approach to automated cellular identification. The success of this methodology has
been driven by its ability to examine large numbers of cells, measure fluorescence
quantitatively, and combine several cellular probes in a single specimen. New applications
are being developed that will not only reveal a cell’s identity, but define its function at the
same time. The probes now available (e.g., from Molecular Probes) consist not only of
antibodies, but also of substrates within the cell itself: molecular probes, lectins, and
ion-sensitive substrates.
Information about the nature of conjugated probes, with particular reference to antibodies,
is presented in UNIT 4.1. The unit discusses techniques for ascertaining the optimal titer for
individual, dual, and multiple antibodies used for simultaneous phenotyping, and also
stresses the importance of quality control in making batches of antibody for routine use.
Methods used to conjugate antibodies to ensure optimal fluorochrome/protein ratios are
discussed in detail in UNIT 4.2. UNIT 4.3 describes the physicochemical structures of a number
of commonly used nucleic acid probes and provides an excellent background for other
protocols in this manual that employ these probes. This unit should be considered
preliminary reading for any studies involving nucleic acid probes. UNIT 4.4 discusses
molecular probes in general, providing considerable background detail for a number of
probes found in the protocols of Chapter 9. For example, probes for hydrogen peroxide
determination (UNIT 9.7), calcium indicators (UNIT 9.8), pH indicators (UNIT 9.3), membrane
potential probes (UNIT 9.6), and green fluorescence protein (UNIT 9.5) are all covered by this
unit. The material in UNIT 4.4 provides an excellent basis for understanding the chemical
and physiological interactions of these fluorescent indicators.
Molecular and
Cellular Probes
Contributed by J. Paul Robinson
Current Protocols in Cytometry (2000) 4.0.1-4.0.2
Copyright © 2000 by John Wiley & Sons, Inc.
4.0.1
Supplement 14
UNIT 4.5 takes the nature of fluorescent probes one step further and provides a quantitative
approach to analysis of DNA and RNA probes using spectroscopic techniques. The study
of cellular fluorescence associated with many fluorescent probes is complemented by a
better quantitative understanding of the relationship between the probe and the relevant
organelle, in this case nuclear material.
LITERATURE CITED
Coons, A.H., Creech, H.J., and Jones, R.N. 1941. Immunological properties of an antibody containing a
fluorescent group. Proc. Soc. Exp. Biol. Med. 47:200-210.
Moller, G. 1961. Demonstration of mouse isoantigens at the cellular level by the fluorescent antibody
technique. J. Exp. Med. 114:415-432.
Weissmann, G. 1992. Inflammation: Historical perspective. In Inflammation: Basic Principle and Clinical
Correlates (J.I. Gallin, I.M. Goldstein, and R. Snyderman, eds.) pp. 5-9. Raven Press, New York.
J. Paul Robinson
Introduction
4.0.2
Supplement 14
Current Protocols in Cytometry
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