Flow cytometry: basic principles and applications

15 Pages·2016·1.58 MB·English

by  Aysun Adan

Critical Reviews in Biotechnology
ISSN: 0738-8551 (Print) 1549-7801 (Online) Journal homepage: http://www.tandfonline.com/loi/ibty20
Flow cytometry: basic principles and applications
Aysun Adan, Günel Alizada, Yağmur Kiraz, Yusuf Baran & Ayten Nalbant
To cite this article: Aysun Adan, Günel Alizada, Yağmur Kiraz, Yusuf Baran & Ayten Nalbant
(2016): Flow cytometry: basic principles and applications, Critical Reviews in Biotechnology,
DOI: 10.3109/07388551.2015.1128876
To link to this article: http://dx.doi.org/10.3109/07388551.2015.1128876
Published online: 14 Jan 2016.
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ISSN:0738-8551(print),1549-7801(electronic)
CritRevBiotechnol,EarlyOnline:1–14
!2016Taylor&Francis.DOI:10.3109/07388551.2015.1128876
REVIEW ARTICLE
Flow cytometry: basic principles and applications
Aysun Adan1*, Gu¨nel Alizada2*, Yag˘mur Kiraz1,2*, Yusuf Baran1,2, and Ayten Nalbant2
1FacultyofLifeandNaturalSciences,AbdullahGu¨lUniversity,Kayseri,Turkeyand2DepartmentofMolecularBiologyandGenetics,I_zmirInstituteof
Technology,I_zmir,Turkey
Abstract Keywords
6 Flowcytometry isasophisticated instrumentmeasuring multiplephysical characteristics of a Apoptosis,cytokines,flowcytometer,
1
0 singlecellsuchassizeandgranularitysimultaneouslyasthecellflowsinsuspensionthrougha fluorescence,fluorescent-activatedcell
2
y measuring device. Its working depends on the light scattering features of the cells under sorting,histogram,immunophenotyping,
Ma investigation, which may be derived from dyes or monoclonal antibodies targeting either lightscatter
9 extracellular molecules located on the surface or intracellular molecules inside the cell. This
0 approachmakesflowcytometryapowerfultoolfordetailedanalysisofcomplexpopulationsin
45 ashortperiod of time.Thisreviewcovers thegeneralprinciples andselectedapplications of History
7: flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis Received18March2015
1 Revised12October2015
at and detection of cytokines. Additionally, this report provides a basic understanding of flow Accepted13October2015
] cytometry technology essential for all users as well as the methods used to analyze and
a Publishedonline8January2016
n interpretthedata.Moreover,recentprogressesinflowcytometryhavebeendiscussedinorder
a
olit togiveanopinionaboutthefutureimportanceofthistechnology.
p
o
r
et
M
a Introduction fluorescence probe is proportional to the amount of fluores-
m cent probe bound to the cell or cellular component (Macey,
o Historically, the first developedflowcytometrywasa single-
n 2010).
o parameter instrument detecting only the size of cells.
ut Therearetwodifferenttypesofflowcytometry–namedas
A Currently, highly sophisticated instruments have evolved
d non-sorting and sorting. Non-sorting type can perform light
a withthecapabilityofdetecting14parameterssimultaneously
d scattering and fluorescence emission while the sorting type
si (Wilkerson,2012).Flowcytometryhastheabilitytomeasure
ver theopticalandfluorescence characteristicsofasinglecell or has the ability to sort particles as well. Fluorescent activated
Uni any other particle such as microorganisms, nuclei and cellsorters(FACS)areflowcytometersthathavethecapacity
tosortfluorescent-labeled cellsfromamixedcell population
by [ chromosome preparations in a fluid stream when they pass (Wilkerson, 2012).
ded tfhluroouregshceantligfhetatsuoruersceo(fMathceey,c2e0ll1s0,).dSeirziev,edgrafnruolmarityeitahnedr The main components of flow cytometers and cell sorters
a are basically fluidics, optics (excitation and collection), an
o antibodies or dyes, are also examples of parameters used to
nl electronicnetwork(detectors)andacomputer.Thefluidicsis
w analyze and differentiate the cells (Wilkerson, 2012). The
o responsible for directing liquid containing particles to the
D underlying principle of flow cytometry is related to light
focused light source. The excitation optic focuses the light
scattering and fluorescence emission, which occurs as light
source on the cells/particles while collection optics transmits
from the excitation source (commonly a laser beam) that
the light scatter or fluorescent light of the particle to an
strikes the moving particles (Figure 1). The data obtained
electronic network. The electronic network detects the signal
could give valuable information about biochemical, biophys-
andconvertsthesignalstoadigitaldatathatisproportionalto
ical and molecular aspects of particles. Light scattering is
light intensity and the computer is also required to analyze
directly related to structural and morphological properties of
data (Shapiro, 2004; Wilkerson, 2012).
the cell while fluorescence emission derived from a
Flow cytometry is used in various applications based on
the detection of the membrane, cytoplasmic and nuclear
antigens. Additionally, whole cells and cellular components
*Theseauthorsequallycontributedtothiswork.
such as organelles, nuclei, DNA, RNA, chromosomes,
Addressforcorrespondence:Assist.Prof.Dr.AytenNalbant,Molecular
cytokines, hormones and protein content can also be
ImmunologyandGeneRegulationLaboratory,DepartmentofMolecular
BiologyandGenetics,I_zmirInstituteofTechnology,Urla,I_zmir35430, investigated by flowcytometry. Analysis of cell proliferation
Turkey. Tel: + 90 232 7507317. Fax: + 90 232 7507303. E-mail: and cell cycle, measurements of calcium flux and membrane
[email protected]
potentials are the commonly used examples of methods
Prof.Dr.YusufBaran,DepartmentofMolecularBiologyandGenetics,
I_zmir Institute of Technology, I_zmir 35430, Turkey. Tel: + 90 232 developed for flow cytometry (Wlodkowic et al.,
7507315.Fax:[phone].E-mail:[email protected] 2011a,2013).
2 A. Adan et al. CritRevBiotechnol,EarlyOnline:1–14
of fluidic components. Therefore, the operator must always
ensurethatthefluidicssystemisfreeofairbubblesanddebris
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
energizingelectronstohighenergyorbitalswithhighvoltage
electricity. Photons of light are produced when these
energized electrons fall back into their lower energy orbitals
(Wilkerson,2012).Lightisdeflectedaroundtheedgesofthe
cell after the laser strikes the cells, also called as light
scattering.Twotypesof lightscatteroccurnamedasforward
scatter (FSC) and side scatters (SSC) (Figure 2). The factors
6
1 affecting total light scatter include the membrane, nucleus,
0
2 granularity of the cell, cell shape and surface topography.
y
a Generally, the size of a cell or a particle and its internal
M
9 complexityspecifythetypeofscatter.FSClightisaresultof
0
5 diffraction collected along the same axis as the laser beam.
7:4 Figure1. Theunderlyingworkingprincipleofaflowcytometer. FCS is proportional to cell-surface area or size and suitable
at 1 for detecting particles greater than a given size that makes it
] Herein, we explained the general principles and selected themostcommonlyusedmethodforimmunophenotyping.On
a
an applications of flow cytometry such as immunophenotyping the other hand, SSC light is a measurement of mostly
olit of peripheral blood cells and analysis of apoptosis. Besides, refracted and reflected light, which is collected at approxi-
op we also discussed the basic data presentation and interpret- mately 90 degrees to the laser beam. SSC is proportional to
r
et ation methods as a helpful material to the researchers who is cell granularity or internal complexity as important as the
M
a interesting in flow cytometric analyses. fluorescent light derived from fluorescent-labeled antibodies
m or dyes such as propidium iodide (PI) are reflected at the
o
on Components of a flow cytometer sameangleasSSC.Inordertodifferentiatethecelltypesina
ut heterogeneous population correlated measurements of FCS
A Fluidic system
d and SSC can be used (Reggeti & Bienzle, 2011).
a
d Thefluidicsystemtransportsthecellsfromasolutionthrough There is a variety of laser configurations in flow
si
er the instrument to obtain the data includes two components; cytometersbasedonthetypeoffluorochromesbeingexcited.
v
ni sheath fluid and pressurized lines. Sheath fluid is a diluent The argon laser (a common laser with an excitation
U
(commonly phosphate-buffered saline (PBS)), which is wavelength of 488nm) is used to excite many synthetic
[
by injected into the flow chamber located at the heart of the dyes such as fluorescein isothiocyanate (FITC) and natural
ed instrument by pressurized lines. A pressurized airline also fluorochrome dyes including algae and phytoplanktons,
d
a injects the suspended cells in the sample tube into the flow resulting in the emission of light at a higher wavelength.
o
nl chamber. The sample stream becomes a central core in the Many flow cytometers and sorters have additional lasers
w
o sheathfluidstreamcalledasacoaxialflowthatisbasedona including ultraviolet that excite UV (300–400nm) sensitive
D
pressure difference between the sheath fluid and sample fluorochromes or the red diode which excites fluorochromes
stream(Macey,2010;Wilkerson,2012).Thesamplepressure of the far red (630nm) range (Macey, 2010; Wilkerson,
is always greater than the sheath fluid pressure, making the 2012).
cells align in a single file fashion through the laser beam. Collection optics consists of a collection of the lens to
Therefore, this event allows uniform illumination of a cell collect light emitted from the particle-laser beam interaction
called hydrodynamic focusing. The injection rate of the cells andasystemofopticalmirrorsandfilterstoseparateandthen
intothelaserbeamcanbemanipulatedbytheflowcytometer direct specified wavelengths of the collected light to the
user based on the purpose of the analysis. For instance, high appropriate optical detectors (Reggeti & Bienzle, 2011).
flow rates are common for qualitative measurements such as The specificity of a detector for a particular fluorescent
immunophenotyping of mammalian cells, whereas slow flow dyeisdeterminedbyplacingasuitablefilter thatcanbelong
rates are appropriate for applications requiring higher reso- pass, short pass and band pass filters. Band pass filters allow
lution such as DNA content analysis. The slow flow rate only a narrow range of wavelengths, which is close to the
makesthesizeofthesamplestreamsmallerwhileitincreases emission peak of the fluorescent dye to reach the detector.
theuniformityandaccuracyoftheillumination(Macey,2010; Short pass filters transmit wavelengths of light equal to or
Shapiro, 2004; Wilkerson, 2012). shorter than a specified wavelength whereas long pass filters
The critical parameter for providing proper interception transmit wavelengths of light equal to or longer than a
between particles and the laser beam is the proper operation specified wavelength (Reggeti & Bienzle, 2011).
DOI:10.3109/07388551.2015.1128876 Flow cytometry 3
Figure 2. Light scattering. FSC is propor-
tionaltosizewhileSSCisproportionalto
cellgranularityorinternalcomplexity.
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45 Signal detection and processing Electrostatic cell sorting
7:
at 1 Light signals generated as particles passing through the laser Electrostatic cell sorting is responsible for capturing and
] beam in a fluid stream are converted to voltages by separationofthecellswithpredefinedfeatures.Oncethecells
a
n photodetectors. Two types of detectors, photodiodes (PDs) of interest are collected, theycan be used for furtheranalysis
a
olit and photomultiplier tubes (PMTs), can be preferred based on such as microscopic, biochemical and functional studies. A
op their sensitivity (Reggeti & Bienzle, 2011; Snow, 2004). singleparameterorcombinationofseveralparameterscanbe
r
et Additionally, the photocathodes of PMT have the greater used for cell sorting. To achieve cell sorting, cell sorters are
M
sensitivity as compared to PD although they are known to commonly used. The general principle of flow cell sorters is
a
m convert light into photoelectrons in a more efficient manner. basedontheelectrostaticdeflectionofchargeddroplets,some
o
n Therefore, PDs detect the stronger light signals generated by of which contain cells. In this method, the cells are injected
o
ut FSC, while PMTs are commonly used to detect the weaker through a nozzle to form a stream of regular droplets by
A
d signals generated by SSC and fluorescence (Figure 3) applying a vibration to the nozzle. Then, these droplets pass
a
d (Reggeti & Bienzle, 2011). through one or more laser beams and are charged by a
si
r ThelightsignalscapturedbyPMTorthePDareconverted chargingelectrodeatthesametime.Dropletscanbedeflected
e
v
ni intoaproportionalnumberofelectronstocreateanelectrical from the mainstream based on theirgiven charges. Positively
U
current. The electrical current travels to the amplifier and is charged droplets are deflected toward a platinum plate of
[
by convertedtoavoltagepulse.Themaximumamountofscatter negative charge, negatively charged droplets are deflected
d or fluorescence is obtained as the particle is in the center of toward the positively charged platinum plate and uncharged
e
d
a thebeam.Whentheparticleleavesthebeam,thepulsecomes droplets are collected into a waste container (Figure 4)
o
nl back down to the baseline (Snow, 2004). (Davies, 2010).
w
o Analogsignalmeasuredasananalogquantityisgenerated FACS is a specialized type of flowcytometry providing a
D
aftertheconversionoftheinitiallightsignalintoanelectrical method for sorting a heterogeneous mixture of fluorescent-
current by the detectors. After that, this electrical current is tagged cells into two or more containers, one cell at a time,
amplified by two types of amplifier, which are linear and based on the specific fluorescent characteristics of each cell
logarithmic. The obtained analog signal must be converted (Davies, 2010). In this case, the operator selects the fluores-
into digital signal by an analog to digital converters for cent cell of interest using a computer as they are passing
computer processing. Then,the signals become adigital data through the laser beam. The tagged cells in the drop will be
that can be displayed as plots or histograms. Although the charged,whichwillbeusedtoseparatethecellsintodifferent
data obtained by using the two mentioned amplifiers is collection vessels (Wilkerson, 2012).
exactlythesame,thedistributionslookdifferentbasedonthe
Principles of fluorescence
approach used to convert an analog signal into digital data.
Moreover, dynamic range is another important parameter to A fluorescent compound has a range of specific wavelengths
choose either linear or logarithmic amplification. Linear at which it absorbs light energy. This absorption of light
amplification is used if a limited dynamic range is required causes an electron to rise from a ground state to a higher
like in DNA analysis (a twofold difference in DNA content). energylevel(excitedstate).Theexcitedelectronquicklygoes
Ontheotherhand,logarithmicamplificationisusedifamuch back to its ground state while giving the excess energy as a
broader dynamic range is needed such as analysis of surface photon of light. This transition of energy is called fluores-
marker expression (100–10000 fold) (Snow, 2004). cence (Figure 5) (Ormerod, 2009).
4 A. Adan et al. CritRevBiotechnol,EarlyOnline:1–14
Figure 3. Signal processing and detection.
Componentsofaflowcytometerarealso
indicated.
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op components that otherwise would not be visible. Fluorescent
r
et probes are used in a wide range of applications such as
M
a identification of different cell populations, cell surface
m receptors or intracellular organelles, cell sorting, immuno-
o
n phenotyping, determining nucleic acid content, measuring
o
ut enzyme activity and apoptotic cell populations (Macey,
A
d 2010).
a
d The important features of a fluorochrome include an
si
er absorptionspectrumatwhichafluorescentcompoundcanbe
v
ni excitedandarangeofemittedwavelengthscalleditsemission
U
spectrum.Theemissionwavelengthofanyfluorochromewill
[
by always be longer than its excitation wavelength (Ormerod,
ed 2009).Thedifferencebetweenthemaximainthewavelengths
d
a ofabsorption and emission is known as the Stoke’s shift that
o
nl determineshowgoodafluorochrome.Thehigher theStoke’s
w
o shift means, the greater the separation between the exciting
D
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
totalphotonsof lightbeingabsorbedbythefluorochromeare
related to the wavelength of excitation. For example, FITC
absorbsthelightwithintherangeof400–550nm,however,it
gives maximum absorbance near 490nm at which more
photons are absorbed. Therefore, the fluorescence emission
will be more intense. These optimal conditions are termed
Figure4. Sortingcellsbydropletdeflection.
maximal absorbance and maximal emission wavelengths
(Ormerod, 2009). In fluorescence detection, a positive
signal is observed against a negative background, which
Fluorochromes and their properties
makes this technique very sensitive. In this technique,
Thenumberofintrinsicallyfluorescentcompoundsinthecell multiparametric analysis of the cells is quite possible due to
is limited as well as its provided information. Therefore, the the detection of up to 14 compound fluorescing at different
cells are usually stained with fluorescent probes called wavelengths. The majority of applications provide the usage
fluorochromes that are able to show the presence of of up to four fluorochromes (Ormerod, 2009).
DOI:10.3109/07388551.2015.1128876 Flow cytometry 5
Figure5.Fluorescencegeneration.
fluorochrome is an important issue and depends on the laser
to be used. If an argon-ion laser including flow cytometer is
6
1 goingtobeused,thefirstchoicewillbeFITC,sincethereare
0
2 numerousFITC-labeledantibodiesavailable.PEisthesecond
y
a choice of color for a similar reason.
M
9 FITC easily reacts with the amino groups on the lysine
0
5 residues in the protein and produces moderately stable
4
7: conjugates. FITC (excitation/emission maxima approx 495/
at 1 520nm) is a good fluorochrome for single-color staining
] since its maximum absorbance near 490nm. On the other
a
n hand, its emissionwith longer wavelengths makesit inappro-
a
olit priate for multicolor applications. Moreover, its fluorescence
op ishighlypH-sensitiveandsubjectedtophotobleachingwitha
r
et high rate. To solve these problems, various FITC derivatives
M
a have been developed such as AlexaTM series with greater
m photostability and increased fluorescence (Macey, 2010).
o
n PE (excitation/emission maxima 495565/578nm) and
o
ut APC (excitation/emission maxima 650/660nm) are called
A
d phycobiliproteins that are the components of photosynthetic
a
d systems. They have good light absorption and high fluores-
si
er cenceintensities.Althoughtheirfluorescenceisabout30-fold
v
ni greater than that of fluorescein, in practice, cells labeled
U
with phycobiliprotein antibodies have fluorescence
[
by intensities between five- and 10-fold greater than those
ed labeledwithFITC-labeledantibody.Althoughusinganargon
d
a laser excites FITC and PE, the excitation of APC needs
o
nl helium–neon laser due to its higher (650nm) absorption
w
Figure6. Theabsorption(greenline)andemission(blueline)spectraof
o maxima. The major drawback of using phycobiliproteins is
D a fluorochrome. The difference between the peak wavelengths of the
related to their higher molecular weight, causing steric
absorptionandemissionspectraisknownastheStokesshift.Thehigher
the Stoke’s shift means the greater the separation between the exciting changes when conjugated to proteins. They can also give
andtheemittedlight(top). higher backgrounds if the cells are not washed properly
(Telford, 2015).
The development of tandem dyes, containing two fluoro-
Fluorochromes used in flow cytometry
chromes, has increased the number of labeled proteins to be
Fluorochromes used in flow cytometry are classified into used. Examples include conjugates of PE and APC with
severalgroupsincludingfluorochromesusedtolabelproteins various cyanine dyes, for instance, PE-Cy(cid:2)5 and APC-Cy7
covalently, fluorochromes for nucleic acids and reporter (Hulspas et al., 2009). In tandem dyes, when the first dye is
molecules. excitedandreachesitsmaximalabsorbance,ittransfersallits
In order to label proteins covalently, the probe is energy to the second dye located in close proximity. As a
commonly selected as an antibody. However, other proteins result,thissecondfluorochromeisactivatedandproducesthe
such as a lectin, hormone, avidin or streptavidin, or even fluorescence emission. This process is called fluorescence
c-DNA may be labeled using various fluorochromes. The resonance energy transfer (FRET). It is a good way to obtain
most widely used fluorochromes for labeling antibodies higher stokes shifts that increase the number of colors
include FITC, phycoerythrin (PE) and allophycocyanin analyzed from a single laser wavelength (Leavesley et al.,
(APC) (Ormerod, 2009). The selection of the most suitable 2013).
6 A. Adan et al. CritRevBiotechnol,EarlyOnline:1–14
Fluorochromes used to stain nucleic acids in flow interestandtofindoutmoreaboutyourcells.Thismethodis
cytometry have some main applications including DNA called ‘‘gating’’ in flow cytometry as well as a gate can be
quantification, the evaluation of membrane integrity and defined as one or more regions combined. A region can be
measurement of ploidy and cell cycle analysis. At present, defined as a set of points carefully selected by the user that
there are many dyes that can stain both RNA and DNA such determinesanareaonagraph.Severalregionscanbedefined
as ethidium bromide (EB) and PI. It is important to remove onthesamegraph.Theseparameterscanprovideyouwithan
RNA with RNase when quantifying DNA. EB and PI opportunity to eliminate results from the unwanted particles
intercalate between the bases in double-stranded nucleic such as dead cells and debris. The most common application
acids. PI is excited by blue light and gives red fluorescence. ofgatingstrategyistouseFSCandSSCplots(Figure8).The
PI can be used in combination with FITC for simultaneous different physical characteristics of white blood cells, gran-
measurement of antibody binding and DNA content if an ulocytes, monocytes and lymphocytes, allow them to be
argon laser is tuned to 488nm. 7-Aminoactinomycin D distinguished from each other.
(7-ADD) characterized by its preferential binding to GC-rich
regionsofDNAisgenerallyremovedbylivecellsandusedto Gates and regions
stain DNA of dead cells. It can be excited by an argon laser
A subset of data can be defined by gating methods.
and used with FITC- and PE-labeled antibodies. There are
Additionally, a gate is a numerical or graphical boundary
also UV-excited dyes such as Hoechst and DAPI that bind to
6 that can be used to define the characteristics of particles for
1 AT-rich regions of DNA. Although they are membrane
0 furtheranalysis.Forexample,inabloodsamplecontaininga
2 permeable dyes, dead cells can uptake them more easily
y mixed population of cells, you might want to restrict
a (Telford et al., 2011; Wlodkowic et al., 2011b).
M your analysis to only the lymphocytes. Based on FSC or
9 Reporter molecules are also used in flow cytometric cellsize,agatecanbesetontheFSCversusSSCplotforthe
0
5 analysis. For instance, green fluorescent protein (GFP) is analysis of lymphocytes. The resulting display would reflect
4
7: excited at 488nm and fluorescent green. GFP and its mutant the fluorescence properties of only the lymphocytes
at 1 forms are used in various applications such sorting of GFP (Figure 9). The data can be displayed by several different
a] transfected cells (Telford et al., 2012). Another reporter plot types. These range from histograms to 2-D plots such as
n molecule is JC-1 dye used to measure mitochondrial mem-
a dot plots, contour and density plots, to 3-D plots such as a
olit brane potential. JC-1 dye accumulates in mitochondria of tomogram plot.
op healthycellsbyformingredJC-1aggregateswithemissionat
r
Met 590nm (Wlodkowic et al., 2011b). In addition, there are Single parameter analysis
a several other reporter molecules to detect several changes in
m experimental cells such as production of the reactive oxygen A histogram is a single parameter plot where the x-axis
o
n species, measurement of intracellular calcium ions and represents the parameter’s signal value in channel numbers
o
ut changes in intracellular pH (Macey, 2010). and the y-axis represents the number of events per channel
A
d number. Each event placed in the channel corresponds to its
da Fluorescence compensation signal value. Signals with identical intensities accumulate in
rsi the same channel. Brighter signals are displayed in channels
ve Eachfluorochromehasawideemissionspectrumresultingin to the right of the dimmer signals. Ideally, flow cytometry
ni some overlap between the fluorochromes when multiple
U willproduceasingledistinctpeak(Figure10).However,flow
y [ fluorochromes are used. This situation is called spectral analysis is sometimes marked on a mixed population of cells
d b overlap and being exemplified as the emission spectra for resulting in several peaks on the histogram.
de FITCandPEareoverlappedatthesamewavelengthsatwhich
a
o some of the light emitted byfluoresceinwill be pass through
nl
w the filter used for PE. This spectral overlap is corrected by Two parameter analysis
o
D subtractingafractionoftheFITCsignalfromthePEsignalor
Alternate methods of data display are available for two-
viceversa.Thisprocessisnamedascolorcompensation,that
parameter plots. These are graphs that display two measure-
also calculates how much interference (as a percent) a
mentparameters,oneonthex-axisandoneonthey-axisand
fluorochrome will have a channel that was not assigned
the cell count as a density (dot) plot or contour map. A dot
specifically to measure it (Roederer, 2001). In order to
plot provides a two-parameter display of data and each dot
determine how much compensation should be applied to the
represents one or more events (particles) while density
dataset, some control readings must be performed first. One
plot displays two parameters as a frequency distribution
of the fluorochromes should be read on itself and then the
(Figure 11). The color is used to code the different
percentofitsdetectabletotalemissioninthechannelspecific
frequenciesofeventsindensityplot(Figure12).Thecontour
for second fluorochrome is determined. This procedure
plot is the same as a density plot, except the density of the
should be repeated for a second fluorochrome as well
cells is displayed as contour lines.
(Figure 7) (Ormerod, 2009).
A quadrant marker divides two-parameter plots into four
sections to discriminate populations as negative, single
Data analysis
positive or double positive. The upper-right quadrant of the
Flowcytometrydataanalysishasbeenconsideredasthemost plot indicates cells positive for both fluorescent marker or
critical parameter for biological experiments. The major double positive. The lower-left quadrant displays events that
principlesofdataanalysisaretoselectivelyshowthecells of are negative for both markers. The upper-left quadrant of the
DOI:10.3109/07388551.2015.1128876 Flow cytometry 7
Figure7. ExplanationoffluorescencecompensationbyusingtwoimaginaryfluorochromesAandB.AismeasuredintheFL-1channelwhileBisin
FL-2channel.OrangepartindicatestheproportionofBthatoverlapsintotheFL-1channel.BluepartindicatestheproportionofAthatentersintothe
FL-2channel.
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si Figure 8. Analysis of freshly isolated human peripheral blood mono-
ver nuclear (PBMCs) cells using SSC/FSC. Separation of PBMCs from Figure 9. Rectangular gates. Peripheral blood mononuclear cells
ni wholebloodismostcommonlyperformedusingFicoll(Bo¨yum,1968). (PBMCs) are plotted as SSC on the y-axis versus CD14 fluorescence
U FSC correlates with the cell volume while SSC correlates with a onthex-axis.FreshlyisolatedhumanPBMCswerewashedandlabeled
[
y granularity of the cell. Neutrophil, monocytes and lymphocyte popula- with CD14 monoclonal antibody (BioLegend, San Diego, CA) for cell
d b tions were identified with their light scattering characteristics alone. A surfaceantigenCD14.Thelabeledcells wereanalyzedbyAttuneflow
de totalof30000eventswereacquiredforanalysis. cytometry. Rectangular gate indicates CD14 positive monocytes in
oa PBMCpopulation.
nl
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Do plot indicates cells only positive for the y-axis parameter.
The lower-right quadrant contains events that are positive for cell surface staining. To obtain the best result of staining,
protocols should be optimized in terms of antibody concen-
the x-axis marker but negative for the y-axis fluorescent
tration, incubation time and temperature for each flow
marker (Figure 13).
cytometry experiment.
Common protocols Cell surface antigens either on cultured or freshly isolated
cells can also be stained. Freshly isolated peripheral blood
Flow cytometry is a powerful technology to study different
mononuclear cells (PBMCs) were labeled with the selected
types of cells and their functions. The most common cell
combination of cell surface antibodies including anti-CD3,
staining principles for identifying and investigating functions
anti-CD4 and anti-CD14 (BD Biosciences, Franklin Lakes,
of a single cell are surface and intracellular staining are
NJ) for 15 min at 4(cid:2)C in the dark. Then, cells were washed
described in detailed below.
two times by centrifugation at 400g for 5 min and
resuspend in the ice-cold PBS. Then, cells were analyzed
Cell surface staining
by flow cytometry (Figures 13 and 14). Attune Acoustic
Cell surface antigens localized on the plasma membrane of Flow Cytometer (Life Technologies, Carlsbad, CA) has been
the cells are the most common proteins to use for flow used to acquire all of the samples and data presented in
cytometry to identify and characterize the cell types. Plasma this review were analyzed by Attune Cytometer Software
membrane proteins are easily accessible to the antibody for (Life Technologies, Carlsbad, CA) version 2.1.0.
8 A. Adan et al. CritRevBiotechnol,EarlyOnline:1–14
Figure10. Asingle-parameterhistogram.Thex-axisrepresentstheparameter’ssignalvalueinchannelnumbers(Count)andthey-axisrepresentsthe
16 numberofeventsperchannelnumber(IL17positivecells).Na¨ıveCD4+TcellswerestimulatedwithTh17cultureconditionduring7thd.Thecells
0 werefixedwithFixationBufferandpermeabilizedwithPermeabilization/WashBufferI(BioLegend).Both(A)unstimulatedand(B)stimulatedcells
2
y were stained with Anti-Human IL-17A Fluorescein-conjugated Antibody (BioLegend). Finally, cells were washed and analyzed with Attune flow
a
M cytometry.
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o Figure11. DotplotofPBMC.CellsubpopulationsbasedonFSCversus
D
SSCwereanalyzedbyflowcytometer.CorrelatedmeasurementsofFSC Figure12. DensityplotsofastimulatednaiveCD4+Tcells.Isolationof
andSSCcanallowcellpopulationtobedistinguishedfromeachother naiveCD4+Thelper cellsfrom PBMCsusingtheNaiveCD4+T Cell
andfromcellularcontaminants.Deadcellshavehighersidescatterand Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive
lowerforwardscatter thanlivingcells. CD4TcellswerestimulatedwithTh17polarizingconditionsfor7d.It
showsallthecellswherethedifferentcolorsrepresentthedensityofthe
cellsatagivenposition.
Intracellular antigen staining
simultaneously at the single-cell level by flow cytometry to
Intracellular proteins of cells can also be detected by flow gain more information about the specific cell.
cytometry. To stain the intracellular proteins, cells are fixed Bcl-2oractivecaspase-3stainingprotocolsweredescribed
with formaldehyde to stabilize the cell membrane antigens here as an example of intracellular protein staining. Cultured
and then permeabilized with detergent or alcohol to allow or freshly isolated cells were harvested, washed with cold
antibodiestoenterintothecell.Itisimportanttofocusonthe PBS and fixed with Cytofix/Cytoperm solution (Pharmingen
location of proteins that will be stained. The presence of containingPBSwith4%paraformaldehydeand0.1%saponin
proteinseitherincytosolornucleusofthecellwilldictatethe for 30 min on ice. Cells were then washed with wash buffer
protocol and buffer system that will be used to get optimum including PBS plus0.1%sodiumazide and1.0%fetalbovine
staining of a protein of interest. It is also possible that cell serum (FBS). Cells were re-suspended and permeabilized in
surface and intracellular staining protocols can be used permeabilization buffer containing PBS with 1% FBS, 0.1%
DOI:10.3109/07388551.2015.1128876 Flow cytometry 9
assessment of apoptosis in cells have been described
including measuring plasma membranes changes, detection
of active caspase-3 and DNA fragmentation.
Cell viability
Themeasurementofcellviabilityplaysanimportantroleinall
kindsofcells.Viabilityanalysiscanprovideinformationabout
theviable(live)andnon-viable(dead)cells.Non-viablecellsin
your samples can non-specifically bind to your antibodies
resultinginfalsepositivesandultimatelyinaccurateresults.The
detection of cell viability by flow cytometry is based on a
simple yet powerful principle (Wlodkowic et al., 2012). The
viabilityofcellscaneasilybedeterminedinflowcytometryby
adding one of DNA binding dyes at a relatively low concen-
trationtoapopulationofcells.Discriminationbetweenliveand
deadcellsinflowcytometricanalysiscanbecarriedoutbased
6 theusageofseveraldifferentfluorochromesincluding7-amino
1
0 actinomycin D (7-AAD) (Figure 17), PI and DAPI.
2 Figure 13. Density plot quadrants. Freshly isolated human peripheral
y bloodmononuclearcellswerelabeledwithCD4andCD3antibodiesfor
a
M cell surface antigens (BioLegend). The labeled cells were analyzed by Detection of plasma membrane changes
9 Attune flow cytometry. Cells were gated on lymphocytes. The x-axis
5 0 represents CD3 positive and the y-axis represents CD4 positive cells. Apoptosis is characterized by a variety of molecular and
4 Upper rightquadrantshowsCD3andCD4doublepositivecells. morphological changes. One of the earliest events in an
7:
1 apoptotic cascade is the alteration in the plasma membrane.
] at The membrane phosphatidylserine is translocated from the
a
n sodiumazideand0.1%saponinfor10minonice.Then,cells inner to the outer leaflet without affecting the plasma
a
olit were stained for 20 min at 4(cid:2)C in the dark with monoclonal membrane integrity, which leads to detect the culmination
p antibodies specific for intracellular antigens such as Bcl-2 of apoptosis by the loss of plasma membrane integrity
o
etr (Figure 15) or active caspase-3 (Figure 16). Following two (Vermes et al., 1995).
M
times washing, cells were analyzed by flow cytometry. CellswerewashedandlabeledwithAnnexinVand7-AAD
ma in the presence of Ca++ binding buffer (Becton Dickinson,
no Applications of flow cytometry Mountain View, CA) for 30 min and analyzed by flow
o
Aut Phenotypic characterization of blood cells cytometry within 1 h in order to measure the plasma
d membrane changes Annexin V-positive, 7-AAD-negative
da Immunophenotypingorphenotypiccharacterizationofcellsis cells were defined as early apoptotic cells with an
rsi theidentificationandquantificationofaspecificcellgroupin intact cell membrane with externalized phosphatidylserine
e
v themixedpopulationusingflowcytometrye.g.immunecells (Figure 18). In contrast, Annexin V-positive and 7-AAD-
ni
U of the blood. Specific cell surface membrane proteins, which positive cells were defined as dead cells.
[
y are known as markers, can be detected by using antibodies.
b
d These specific antigens can be expressed on more than one Detection of active caspase-3 activity
e
d cell type.
oa Another molecular hallmark of apoptosis is the activation of
nl Therefore, immunophenotyping of the cells can be caspases. Caspases are inactive enzymes and must become
w accomplished by staining of a single cell simultaneously
o activeduringapoptosis.Oneoftheeffectorcaspasesactivated
D with two or more antibodies. Phenotypic characterization of
at the late apoptosis stage is caspase-3. Active caspases-3
blood cells is seen in Figure 17. Human PBMCs were first
initiates series of events that ultimately kill a cell (Lessene
isolated by Ficoll-Hypaque density gradient centrifugation
et al., 2008; Wlodkowic et al., 2012). The active caspase-3
(Bo¨yum,1968).Then,PBMCswerelabeledwiththeselected
can be detected and analyzed with flow cytometry
combination of cell surface antibodies including anti-CD3,
(Figure 16).
anti-CD4 and anti-CD14 (BD Biosciences) (Figure 14). Cell
surfacelabelingwascarriedoutasdescribedin‘‘CellSurface
Staining’’ section. Cell surface staining section and labeled Detection of mitochondrial proteins
cells were analyzed by flow cytometry.
Bcl2isahumanproto-oncogenelocatedinthemembranesof
the endoplasmic reticulum, nuclear envelope and inthe outer
Measurement of apoptosis markers
membrane of mitochondria. Preclinical studies demonstrated
Rapidandquantitativemeasurementofapoptoticcellscanbe that Bcl2 is anti-apoptotic intracellular protein and the
detectedbyflowcytometry.Themethodsfor thedetectionof decreased Bcl2 level indicates progression of apoptosis
cells undergoing apoptosis (programmed cell death) should (Wlodkowic et al., 2012). In order to detect Bcl2 activity,
detect apoptotic properties of cells rapidlyand allow them to cells were fixed and permeabilized. Then fluorescence-
keep their natural state in order to obtain the most reliable conjugated Bcl2 antibody was added into cells and analyzed
results. Many different flow cytometric methods for the by flow cytometry (Figure 15).