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METHODS IN MOLECULAR BIOLOGY
TM
METHODS IN MOLECULAR BIOLOGY
TM
Volume 263
Flow Cytometry
Protocols
S
ECOND
E
DITION
Flow Cytometry
Protocols
S
ECOND
E
DITION
Edited by
Edited by
Teresa S. Hawley
Robert G. Hawley
Teresa S. Hawley
Robert G. Hawley
1
Flow Cytometry
An Introduction
Alice L. Givan
Summary
A flow cytometer is an instrument that illuminates cells (or other particles) as they flow indi-
vidually in front of a light source and then detects and correlates the signals from those cells that
result from the illumination. In this chapter, each of the aspects of that definition will be
described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the
use of fluidics to guide the cells individually past the illuminating beam, the types of signals
emitted by the cells and the detection of those signals, the conversion of light signals to digital
data, and the use of computers to correlate and analyze the data after they are stored in a data file.
The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chap-
ter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts
into the field. Other chapters in this book will provide more details, more references, and even
some controversy about specific topics.
Key Words
Flow cytometry, fluidics, fluorescence, laser.
1. Introduction
An introductory chapter on flow cytometry must first confront the difficulty
of defining a flow cytometer. The instrument described by Andrew Moldavan in
1934
(1)
is generally acknowledged to be an early prototype. Although it may
never have been built, in design it looked like a microscope but provided a cap-
illary tube on the stage so that cells could be individually illuminated as they
flowed in single file in front of the light emitted through the objective. The
signals coming from the cells could then be analyzed by a photodetector
attached in the position of the microscope eyepiece. Following work by Coul-
From:
Methods in Molecular Biology: Flow Cytometry Protocols, 2nd ed.
Edited by: T. S. Hawley and R. G. Hawley © Humana Press Inc., Totowa, NJ
1
2
Givan
ter and others in the next decades to develop instruments to count particles in
suspension (
see
refs.
2–5)
,adesign was implemented by Kamentsky and
Melamed in 1965 and 1967
(6
,
7)
to produce a microscope-based flow cytome-
ter for detecting light signals distinguishing the abnormal cells in a cervical
sample. In the years after publication of the Kamentsky papers, work by
Fulwyler, Dittrich and Göhde, Van Dilla, and Herzenberg (
see
refs.
8–11)
led to
significant changes in overall design, resulting in a cytometer that was largely
similar to today’s cytometers. Like today’s cytometers, a flow cytometer in 1969
did not resemble a microscope in any way but was still based on Moldavan’s
prototype and on the Kamentsky instrument in that it illuminated cells as they
progressed in single file in front of a beam of light and it used photodetectors to
detect the signals that came from the cells (
see
Shapiro
[12]
and Melamed
[13
,
14]
for more complete discussions of this historical development). Even
today, our definition of a flow cytometer involves an instrument that illuminates
cells as they flow individually in front of a light source and then detects and cor-
relates the signals from those cells that result from the illumination.
In this chapter, each of the aspects in that definition are described: the cells,
methods to illuminate the cells, the use of fluidics to make sure that the cells
flow individually past the illuminating beam, the use of detectors to mea-
sure the signals coming from the cells, and the use of computers to correlate the
signals after they are stored in data files. As an introduction, this chapter can
be read as a brief survey; it can also be read as a gateway with signposts into
the field. Other chapters in this book (and in other books [e.g.,
refs.
12
,
15–24)
provide more details, more references, and even some controversy concerning
specific topics.
2. Cells (or Particles or Events)
Before discussing “cells,” we need to qualify even that basic word. “Cytome-
ter” is derived from two Greek words, “
”, meaning container, receptacle,
or body (taken in modern formations to mean cell), and “
κντοζ
”, meaning
measure. Cytometers today, however, often measure things other than cells. “Par-
ticle” can be used as a more general term for any of the objects flowing through
a flow cytometer. “Event” is a term that is used to indicate anything that has
been interpreted by the instrument, correctly or incorrectly, to be a single parti-
cle. There are subtleties here; for example, if the cytometer is not quick enough,
two particles close together may actually be detected as one event. Because
most of the particles sent through cytometers and detected as events are, in fact,
single cells, those words are used here somewhat interchangeably.
Because flow cytometry is a technique for the analysis of individual parti-
cles, a flow cytometrist must begin by obtaining a suspension of particles. His-
torically, the particles analyzed by flow cytometry were often cells from the
µετρον
Flow Cytometry: An Introduction
3
blood; these are ideally suited for this technique because they exist as single
cells and require no manipulation before cytometric analysis. Cultured cells or
cell lines have also been suitable, although adherent cells require some treat-
ment to remove them from the surface on which they are grown. More recently,
bacteria
(25
,
26)
,sperm
(27
,
28)
, and plankton
(29)
have been analyzed. Flow
techniques have also been used to analyze individual particles that are not cells
at all (e.g., viruses
[30]
, nuclei
[31]
, chromosomes
[32]
,DNA fragments
[33]
,
and latex beads
[34]
). In addition, cells that do not occur as single particles
can be made suitable for flow cytometric analysis by the use of mechanical
disruption or enzymatic digestion; tissues can be disaggregated into individual
cells and these can be run through a flow cytometer. The advantage of a method
that analyzes single cells is that cells can be scanned at a rapid rate (500 to
>5000 per second) and the individual characteristics of a large number of cells
can be enumerated, correlated, and summarized. The disadvantage of a single-
cell technique is that cells that do not occur as individual particles will need to
be disaggregated; when tissues are disaggregated for analysis, some of the char-
acteristics of the individual cells can be altered and all information about tissue
architecture and cell distribution is lost.
In flow cytometry, because particles flow in a narrow stream in front of a
narrow beam of light, there are size restrictions. In general, cells or particles
must fall between approx 1
m in diameter. Special cytome-
ters may have the increased sensitivity to handle smaller particles (such as DNA
fragments
[33]
or small bacteria
[35]
) or may have the generous fluidics to
handle larger particles (such as plant cells
[36]
). But ordinary cytometers will,
on the one hand, not be sensitive enough to detect signals from very small par-
ticles and will, on the other hand, become obstructed with very large particles.
Particles for flow cytometry should be suspended in buffer at a concentration
of about 5
µ
m and approx 30
µ
10
6
/mL. In this suspension, they will flow through the
cytometer mostly one by one. The light emitted from each particle will be
detected and stored in a data file for subsequent analysis. In terms of the emit-
ted light, particles will scatter light and this scattered light can be detected.
Some of the emitted light is not scattered light, but is fluorescence. Many par-
ticles (notably phytoplankton) have natural background (auto-) fluorescence
and this can be detected by the cytometer. In most cases, particles without
intrinsically interesting autofluorescence will have been stained with fluorescent
dyes during preparation to make nonfluorescent compounds “visible” to the
cytometer. A fluorescent dye is one that absorbs light of certain specific colors
and then emits light of a different color (usually of a longer wavelength). The
fluorescent dyes may be conjugated to antibodies and, in this case, the fluo-
rescence of a cell will be a readout for the amount of protein/antigen (on the
cell surface or in the cytoplasm or nucleus) to which the antibody has bound.
×
10
5
to 5
×
4
Givan
Some fluorochrome-conjugated molecules can be used to indicate apoptosis
(37)
. Alternatively, the dye itself may fluoresce when it is bound to a cellular
component. Staining with DNA-sensitive fluorochromes can be used, for exam-
ple, to look at multiploidy in mixtures of malignant and normal cells
(31)
; in
conjunction with mathematical algorithms, to study the proportion of cells in
different stages of the cell cycle
(38)
; and in restriction-enzyme-digested mate-
rial, to type bacteria according to the size of their fragmented DNA
(39)
. There
are other fluorochromes that fluoresce differently in relation to the concentra-
tion of calcium ions
(40)
or protons
(41
,
42)
in the cytoplasm or to the poten-
tial gradient across a cell or organelle membrane
(43)
. In these cases, the
fluorescence of the cell may indicate the response of that cell to stimulation.
Other dyes can be used to stain cells in such a way that the dye is partitioned
between daughter cells on cell division; the fluorescence intensity of the cells
will reveal the number of divisions that have occurred
(44)
. Chapters in this
book provide detailed information about fluorochromes and their use. In addi-
tion, the Molecular Probes (Eugene, OR) handbook by Richard Haugland is
an excellent, if occasionally overwhelming, source of information about fluor-
escent molecules.
The important thing to know about the use of fluorescent dyes for staining
cells is that the dyes themselves need to be appropriate to the cytometer. This
requires knowledge of the wavelength of the illuminating light, knowledge of
the wavelength specificities of the filters in front of the instrument’s photode-
tectors, and knowledge of the absorption and emission characteristics of the
dyes themselves. The fluorochromes used to stain cells must be able to absorb
the particular wavelength of the illuminating light and the detectors must have
appropriate filters to detect the fluorescence emitted. For the purposes of this
introductory chapter, we now assume that we have particles that are individu-
ally suspended in medium at a concentration of about 1 million/mL and that
they have been stained with (or naturally contain) fluorescent molecules with
appropriate wavelength characteristics.
3. Illumination
In most flow cytometers, fluorescent cells are illuminated with the light from
a laser. Lasers are useful because they provide intense light in a narrow beam.
Particles in a stream of fluid can move through this light beam rapidly; under
ideal circumstances, only one particle will be illuminated at a time, and the
illumination is bright enough to produce scattered light or fluorescence of
detectable intensity.
Lasers in today’s cytometers are either gas lasers (e.g., argon ion lasers or
helium–neon lasers) or solid-state lasers (e.g., red or green diode lasers or the
relatively new blue and violet lasers). In all cases, light of specific wavelengths
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