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ISSN: 0738-8551 (print), 1549-7801 (electronic)
Crit Rev Biotechnol, 2017; 37(2): 163–176
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/07388551.2015.1128876
REVIEW ARTICLE
Flow cytometry: basic principles and applications
Aysun Adan
1
*,Gu¨ nel Alizada
2
*, Yag
˘
mur Kiraz
1,2
*, Yusuf Baran
1,2
, and Ayten Nalbant
2
1
Faculty of Life and Natural Sciences, Abdullah Gu¨l University, Kayseri, Turkey and
2
Department of Molecular Biology and Genetics,
_
Izmir Institute of
Technology,
_
Izmir, Turkey
Abstract
Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a
single cell such as size and granularity simultaneously as the cell flows in suspension through a
measuring device. Its working depends on the light scattering features of the cells under
investigation, which may be derived from dyes or monoclonal antibodies targeting either
extracellular molecules located on the surface or intracellular molecules inside the cell. This
approach makes flow cytometry a powerful tool for detailed analysis of complex populations in
a short period of time. This review covers the general principles and selected applications of
flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis
and detection of cytokines. Additionally, this report provides a basic understanding of flow
cytometry technology essential for all users as well as the methods used to analyze and
interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order
to give an opinion about the future importance of this technology.
Keywords
Apoptosis, cytokines, flow cytometer,
fluorescence, fluorescent-activated cell
sorting, histogram, immunophenotyping,
light scatter
History
Received 18 March 2015
Revised 12 October 2015
Accepted 13 October 2015
Published online 8 January 2016
Introduction
Historically, the first developed flow cytometry was a single-
parameter instrument detecting only the size of cells.
Currently, highly sophisticated instruments have evolved
with the capability of detecting 14 parameters simultaneously
(Wilkerson, 2012). Flow cytometry has the ability to measure
the optical and fluorescence characteristics of a single cell or
any other particle such as microorganisms, nuclei and
chromosome preparations in a fluid stream when they pass
through a light source (Macey, 2010). Size, granularity and
fluorescent features of the cells, derived from either
antibodies or dyes, are also examples of parameters used to
analyze and differentiate the cells (Wilkerson, 2012). The
underlying principle of flow cytometry is related to light
scattering and fluorescence emission, which occurs as light
from the excitation source (commonly a laser beam) that
strikes the moving particles (Figure 1). The data obtained
could give valuable information about biochemical, biophys-
ical and molecular aspects of particles. Light scattering is
directly related to structural and morphological properties of
the cell while fluorescence emission derived from a
fluorescence probe is proportional to the amount of fluores-
cent probe bound to the cell or cellular component (Macey,
2010).
There are two different types of flow cytometry – named as
non-sorting and sorting. Non-sorting type can perform light
scattering and fluorescence emission while the sorting type
has the ability to sort particles as well. Fluorescent activated
cell sorters (FACS) are flow cytometers that have the capacity
to sort fluorescent-labeled cells from a mixed cell population
(Wilkerson, 2012).
The main components of flow cytometers and cell sorters
are basically fluidics, optics (excitation and collection), an
electronic network (detectors) and a computer. The fluidics is
responsible for directing liquid containing particles to the
focused light source. The excitation optic focuses the light
source on the cells/particles while collection optics transmits
the light scatter or fluorescent light of the particle to an
electronic network. The electronic network detects the signal
and converts the signals to a digital data that is proportional to
light intensity and the computer is also required to analyze
data (Shapiro, 2004; Wilkerson, 2012).
Flow cytometry is used in various applications based on
the detection of the membrane, cytoplasmic and nuclear
antigens. Additionally, whole cells and cellular components
such as organelles, nuclei, DNA, RNA, chromosomes,
cytokines, hormones and protein content can also be
investigated by flow cytometry. Analysis of cell proliferation
and cell cycle, measurements of calcium flux and membrane
potentials are the commonly used examples of methods
developed for flow cytometry (Wlodkowic et al.,
2011a,2013).
*These authors equally contributed to this work.
Address for correspondence: Assist. Prof. Dr. Ayten Nalbant, Molecular
Immunology and Gene Regulation Laboratory, Department of Molecular
Biology and Genetics,
_
Izmir Institute of Technology, Urla,
_
Izmir 35430,
Turkey. Tel: + 90 232 7507317. Fax: + 90 232 7507303. E-mail:
aytennalbant@iyte.edu.tr
Prof. Dr. Yusuf Baran, Department of Molecular Biology and Genetics,
_
Izmir Institute of Technology,
_
Izmir 35430, Turkey. Tel: + 90 232
7507315. Fax: + 90 232 7507300. E-mail: ybaran@gmail.com
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Herein, we explained the general principles and selected
applications of flow cytometry such as immunophenotyping
of peripheral blood cells and analysis of apoptosis. Besides,
we also discussed the basic data presentation and interpret-
ation methods as a helpful material to the researchers who is
interesting in flow cytometric analyses.
Components of a flow cytometer
Fluidic system
The fluidic system transports the cells from a solution through
the instrument to obtain the data includes two components;
sheath fluid and pressurized lines. Sheath fluid is a diluent
(commonly phosphate-buffered saline (PBS)), which is
injected into the flow chamber located at the heart of the
instrument by pressurized lines. A pressurized airline also
injects the suspended cells in the sample tube into the flow
chamber. The sample stream becomes a central core in the
sheath fluid stream called as a coaxial flow that is based on a
pressure difference between the sheath fluid and sample
stream (Macey, 2010; Wilkerson, 2012). The sample pressure
is always greater than the sheath fluid pressure, making the
cells align in a single file fashion through the laser beam.
Therefore, this event allows uniform illumination of a cell
called hydrodynamic focusing. The injection rate of the cells
into the laser beam can be manipulated by the flow cytometer
user based on the purpose of the analysis. For instance, high
flow rates are common for qualitative measurements such as
immunophenotyping of mammalian cells, whereas slow flow
rates are appropriate for applications requiring higher reso-
lution such as DNA content analysis. The slow flow rate
makes the size of the sample stream smaller while it increases
the uniformity and accuracy of the illumination (Macey, 2010;
Shapiro, 2004; Wilkerson, 2012).
The critical parameter for providing proper interception
between particles and the laser beam is the proper operation
of fluidic components. Therefore, the operator must always
ensure that the fluidics system is free of air bubbles and debris
and is properly pressurized at all times.
Optical system
A flow cytometer has an optical bench that holds the
excitation, which includes the laser and lenses and collection
optics in fixed positions. The lenses are used to shape and
focus the laser beam. Meanwhile, the laser produces light by
energizing electrons to high energy orbitals with high voltage
electricity. Photons of light are produced when these
energized electrons fall back into their lower energy orbitals
(Wilkerson, 2012). Light is deflected around the edges of the
cell after the laser strikes the cells, also called as light
scattering. Two types of light scatter occur named as forward
scatter (FSC) and side scatters (SSC) (Figure 2). The factors
affecting total light scatter include the membrane, nucleus,
granularity of the cell, cell shape and surface topography.
Generally, the size of a cell or a particle and its internal
complexity specify the type of scatter. FSC light is a result of
diffraction collected along the same axis as the laser beam.
FCS is proportional to cell-surface area or size and suitable
for detecting particles greater than a given size that makes it
the most commonly used method for immunophenotyping. On
the other hand, SSC light is a measurement of mostly
refracted and reflected light, which is collected at approxi-
mately 90 degrees to the laser beam. SSC is proportional to
cell granularity or internal complexity as important as the
fluorescent light derived from fluorescent-labeled antibodies
or dyes such as propidium iodide (PI) are reflected at the
same angle as SSC. In order to differentiate the cell types in a
heterogeneous population correlated measurements of FCS
and SSC can be used (Reggeti & Bienzle, 2011).
There is a variety of laser configurations in flow
cytometers based on the type of fluorochromes being excited.
The argon laser (a common laser with an excitation
wavelength of 488 nm) is used to excite many synthetic
dyes such as fluorescein isothiocyanate (FITC) and natural
fluorochrome dyes including algae and phytoplanktons,
resulting in the emission of light at a higher wavelength.
Many flow cytometers and sorters have additional lasers
including ultraviolet that excite UV (300–400 nm) sensitive
fluorochromes or the red diode which excites fluorochromes
of the far red (630 nm) range (Macey, 2010; Wilkerson,
2012).
Collection optics consists of a collection of the lens to
collect light emitted from the particle-laser beam interaction
and a system of optical mirrors and filters to separate and then
direct specified wavelengths of the collected light to the
appropriate optical detectors (Reggeti & Bienzle, 2011).
The specificity of a detector for a particular fluorescent
dye is determined by placing a suitable filter that can be long
pass, short pass and band pass filters. Band pass filters allow
only a narrow range of wavelengths, which is close to the
emission peak of the fluorescent dye to reach the detector.
Short pass filters transmit wavelengths of light equal to or
shorter than a specified wavelength whereas long pass filters
transmit wavelengths of light equal to or longer than a
specified wavelength (Reggeti & Bienzle, 2011).
Figure 1. The underlying working principle of a flow cytometer.
164 A. Adan et al. Crit Rev Biotechnol, 2017; 37(2): 163–176
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Signal detection and processing
Light signals generated as particles passing through the laser
beam in a fluid stream are converted to voltages by
photodetectors. Two types of detectors, photodiodes (PDs)
and photomultiplier tubes (PMTs), can be preferred based on
their sensitivity (Reggeti & Bienzle, 2011; Snow, 2004).
Additionally, the photocathodes of PMT have the greater
sensitivity as compared to PD although they are known to
convert light into photoelectrons in a more efficient manner.
Therefore, PDs detect the stronger light signals generated by
FSC, while PMTs are commonly used to detect the weaker
signals generated by SSC and fluorescence (Figure 3)
(Reggeti & Bienzle, 2011).
The light signals captured by PMT or the PD are converted
into a proportional number of electrons to create an electrical
current. The electrical current travels to the amplifier and is
converted to a voltage pulse. The maximum amount of scatter
or fluorescence is obtained as the particle is in the center of
the beam. When the particle leaves the beam, the pulse comes
back down to the baseline (Snow, 2004).
Analog signal measured as an analog quantity is generated
after the conversion of the initial light signal into an electrical
current by the detectors. After that, this electrical current is
amplified by two types of amplifier, which are linear and
logarithmic. The obtained analog signal must be converted
into digital signal by an analog to digital converters for
computer processing. Then, the signals become a digital data
that can be displayed as plots or histograms. Although the
data obtained by using the two mentioned amplifiers is
exactly the same, the distributions look different based on the
approach used to convert an analog signal into digital data.
Moreover, dynamic range is another important parameter to
choose either linear or logarithmic amplification. Linear
amplification is used if a limited dynamic range is required
like in DNA analysis (a twofold difference in DNA content).
On the other hand, logarithmic amplification is used if a much
broader dynamic range is needed such as analysis of surface
marker expression (100–10 000 fold) (Snow, 2004).
Electrostatic cell sorting
Electrostatic cell sorting is responsible for capturing and
separation of the cells with predefined features. Once the cells
of interest are collected, they can be used for further analysis
such as microscopic, biochemical and functional studies. A
single parameter or combination of several parameters can be
used for cell sorting. To achieve cell sorting, cell sorters are
commonly used. The general principle of flow cell sorters is
based on the electrostatic deflection of charged droplets, some
of which contain cells. In this method, the cells are injected
through a nozzle to form a stream of regular droplets by
applying a vibration to the nozzle. Then, these droplets pass
through one or more laser beams and are charged by a
charging electrode at the same time. Droplets can be deflected
from the mainstream based on their given charges. Positively
charged droplets are deflected toward a platinum plate of
negative charge, negatively charged droplets are deflected
toward the positively charged platinum plate and uncharged
droplets are collected into a waste container (Figure 4)
(Davies, 2010).
FACS is a specialized type of flow cytometry providing a
method for sorting a heterogeneous mixture of fluorescent-
tagged cells into two or more containers, one cell at a time,
based on the specific fluorescent characteristics of each cell
(Davies, 2010). In this case, the operator selects the fluores-
cent cell of interest using a computer as they are passing
through the laser beam. The tagged cells in the drop will be
charged, which will be used to separate the cells into different
collection vessels (Wilkerson, 2012).
Principles of fluorescence
A fluorescent compound has a range of specific wavelengths
at which it absorbs light energy. This absorption of light
causes an electron to rise from a ground state to a higher
energy level (excited state). The excited electron quickly goes
back to its ground state while giving the excess energy as a
photon of light. This transition of energy is called fluores-
cence (Figure 5) (Ormerod, 2009).
Figure 2. Light scattering. FSC is propor-
tional to size while SSC is proportional to
cell granularity or internal complexity.
DOI: 10.3109/07388551.2015.1128876 Flow cytometry 165
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Fluorochromes and their properties
The number of intrinsically fluorescent compounds in the cell
is limited as well as its provided information. Therefore, the
cells are usually stained with fluorescent probes called
fluorochromes that are able to show the presence of
components that otherwise would not be visible. Fluorescent
probes are used in a wide range of applications such as
identification of different cell populations, cell surface
receptors or intracellular organelles, cell sorting, immuno-
phenotyping, determining nucleic acid content, measuring
enzyme activity and apoptotic cell populations (Macey,
2010).
The important features of a fluorochrome include an
absorption spectrum at which a fluorescent compound can be
excited and a range of emitted wavelengths called its emission
spectrum. The emission wavelength of any fluorochrome will
always be longer than its excitation wavelength (Ormerod,
2009). The difference between the maxima in the wavelengths
of absorption and emission is known as the Stoke’s shift that
determines how good a fluorochrome. The higher the Stoke’s
shift means, the greater the separation between the exciting
and the emitted light (Figure 6) (Ormerod, 2009). Since the
color of the exciting and emitting light is different, they can
be separated from one another by using optical filters. The
total photons of light being absorbed by the fluorochrome are
related to the wavelength of excitation. For example, FITC
absorbs the light within the range of 400–550 nm, however, it
gives maximum absorbance near 490 nm at which more
photons are absorbed. Therefore, the fluorescence emission
will be more intense. These optimal conditions are termed
maximal absorbance and maximal emission wavelengths
(Ormerod, 2009). In fluorescence detection, a positive
signal is observed against a negative background, which
makes this technique very sensitive. In this technique,
multiparametric analysis of the cells is quite possible due to
the detection of up to 14 compound fluorescing at different
wavelengths. The majority of applications provide the usage
of up to four fluorochromes (Ormerod, 2009).
Figure 3. Signal processing and detection.
Components of a flow cytometer are also
indicated.
Figure 4. Sorting cells by droplet deflection.
166 A. Adan et al. Crit Rev Biotechnol, 2017; 37(2): 163–176
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Fluorochromes used in flow cytometry
Fluorochromes used in flow cytometry are classified into
several groups including fluorochromes used to label proteins
covalently, fluorochromes for nucleic acids and reporter
molecules.
In order to label proteins covalently, the probe is
commonly selected as an antibody. However, other proteins
such as a lectin, hormone, avidin or streptavidin, or even
c-DNA may be labeled using various fluorochromes. The
most widely used fluorochromes for labeling antibodies
include FITC, phycoerythrin (PE) and allophycocyanin
(APC) (Ormerod, 2009). The selection of the most suitable
fluorochrome is an important issue and depends on the laser
to be used. If an argon-ion laser including flow cytometer is
going to be used, the first choice will be FITC, since there are
numerous FITC-labeled antibodies available. PE is the second
choice of color for a similar reason.
FITC easily reacts with the amino groups on the lysine
residues in the protein and produces moderately stable
conjugates. FITC (excitation/emission maxima approx 495/
520 nm) is a good fluorochrome for single-color staining
since its maximum absorbance near 490 nm. On the other
hand, its emission with longer wavelengths makes it inappro-
priate for multicolor applications. Moreover, its fluorescence
is highly pH-sensitive and subjected to photobleaching with a
high rate. To solve these problems, various FITC derivatives
have been developed such as Alexa
TM
series with greater
photostability and increased fluorescence (Macey, 2010).
PE (excitation/emission maxima 495 565/578 nm) and
APC (excitation/emission maxima 650/660 nm) are called
phycobiliproteins that are the components of photosynthetic
systems. They have good light absorption and high fluores-
cence intensities. Although their fluorescence is about 30-fold
greater than that of fluorescein, in practice, cells labeled
with phycobiliprotein antibodies have fluorescence
intensities between five- and 10-fold greater than those
labeled with FITC-labeled antibody. Although using an argon
laser excites FITC and PE, the excitation of APC needs
helium–neon laser due to its higher (650 nm) absorption
maxima. The major drawback of using phycobiliproteins is
related to their higher molecular weight, causing steric
changes when conjugated to proteins. They can also give
higher backgrounds if the cells are not washed properly
(Telford, 2015).
The development of tandem dyes, containing two fluoro-
chromes, has increased the number of labeled proteins to be
used. Examples include conjugates of PE and APC with
various cyanine dyes, for instance, PE-CyÔ5 and APC-Cy7
(Hulspas et al., 2009). In tandem dyes, when the first dye is
excited and reaches its maximal absorbance, it transfers all its
energy to the second dye located in close proximity. As a
result, this second fluorochrome is activated and produces the
fluorescence emission. This process is called fluorescence
resonance energy transfer (FRET). It is a good way to obtain
higher stokes shifts that increase the number of colors
analyzed from a single laser wavelength (Leavesley et al.,
2013).
Figure 6. The absorption (green line) and emission (blue line) spectra of
a fluorochrome. The difference between the peak wavelengths of the
absorption and emission spectra is known as the Stokes shift. The higher
the Stoke’s shift means the greater the separation between the exciting
and the emitted light (top).
Figure 5. Fluorescence generation.
DOI: 10.3109/07388551.2015.1128876 Flow cytometry 167
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