“THE FULL SPECTRUM”

A BEGINNER’S GUIDE
TO FLOW CYTOMETRY

www.innovabiosciences.com
A BEGINNER’S GUIDE TO FLOW CYTOMETRY
Introduction

Flow Cytometry is a widely used method for cell analysis which, for the novice, can appear daunting due the complexity
of the experimental approach and data analysis. This simple introductory guide has been written with such individuals
in mind. It is intended to provide an overview of:

• Flow Cytometry and its associated applications

• The basic components of the flow cytometer
• The main complexities of flow cytometry
• The benefits of Lightning-Link® - a 30 seconds hands on, simple antibody and protein labelling technique

What is flow cytometry and what can it do?

With respect to cellular analysis, the underlying principle of flow cytometry is that a cell suspension is focussed into a
single cell stream which passes through a light source (typically a laser beam). The scattered and emitted fluorescent
light (if the cells are fluorescently labelled) is subsequently measured using a range of detectors and these measurements
are used to generate multi-parameter data sets that describe the physical characteristics of the cells and their fluorescent
properties. The size and granularity of cells can be identified on the basis of their forward and side light scatter charac-
teristics (FSc and SSc respectively). Their characteristics and/or expression of different proteins can be further defined
by pre-staining with fluorescently-labelled antibodies or molecules that identify cellular components and / or integrity
(viability) or, indeed, fluorescently-labelled proteins.

Fluorescent dyes accept light energy at a given wavelength (excitation) and re-emit at a longer wavelength (emission).
The fluorescent light which is emitted by these dyes when they are excited by the appropriate light source is channelled
via appropriate filters and the signals are collected by an array of detectors. Linking such fluorochromes to an antibody or
using fluorescently-labelled molecules that bind to cellular components allows profiles of proteins that are expressed
by these cells to be identified and characterized by flow cytometry. With appropriate instrumentation, it is possible to
identify particular cell subpopulations on the basis of their physical and / or fluorescent characteristics and isolate or
‘sort’ these.

Some applications of flow cytometry

• DNA/Cell Cycle analysis • Cell viability

• Cell proliferation • Intracellular ionic (e.g. Ca2+) fluxes
• Multicolor phenotyping (cell surface) • Multicolor phenotyping (intracellular)
• Monocyte oxidative burst • Monocyte phagocytosis
• Neutrophil oxidative burst • Neutrophil phagocytosis
• Microbiological analysis • Cell trafficking
• Cellular and antibody or complement-mediated cytotoxicity • Sorting on the basis of morphology (FSC or SSc) and/
or fluorescent characteristics

Flow cytometers can evaluate cells at an extremely rapid rate (up to several tens of thousands
of events (cells) per second) and can detect as few as 500 molecules per cell. The speed
and sensitivity of flow cytometry therefore makes it ideally suited to the analysis of minor
cellular populations.

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY
The Flow Cytometer – Basic Components

The Flow Chamber (also known as fluidics; please see diagram to

Light
 Flow

the right) is at the heart of the instrument system and is designed Source
 Chamber

4p6cal


System

to deliver a single cell stream at the point of measurement. This is
achieved by focussing cells into the centre of a narrow stream of
liquid called sheath fluid such that only single cells pass through
the illuminating beam of light in the flow chamber. Although the Computer
 Light

Detectors

majority of instruments achieve a single cell stream using fluidics,
instruments which achieve this using acoustic focussing are now
being developed.
Sample
The flow rate of sheath fluid will determine how rapidly the stream Sheath

of material will move, and thus reach the laser(s) of the cytom-
eter. In an ideal situation, material will pass through the laser
one-by-one, and thus data from each individual call (‘event’) will
be recorded. This will provide the most accurate results and it is Cells

therefore very important to understand the capabilities of your

cytometer.

Some cytometers have a peak detection rate of 900 events per

second. Should the flow rate deliver cells at a rate which is faster
Scatter &
than the recommended maximum of the instrument being used, Fluorescence
then 2 or more could pass through the laser simultaneously (‘co-
incidental events’). This will only result in only one event being
recorded and a loss of data. Although a warning message such
as a ‘data rate warning’ might occur on the cytometers software, Flow
it is important to understand the consequences should the sheath
flow be set too high.

The light source can be a laser, arc lamp or light emitting diode (LED). Arc lamps (e.g. mercury, xenon-mercury) are
bright and powerful light sources that emit light at multiple wavelengths. They are typically found in light microscopes.
Although this broad excitation spectrum is an advantage in some instances, it can also be a disadvantage. A far more
common light source in flow cytometers is the laser. These provide a bright and coherent light source at well-defined and
specific narrow wavelengths. A number of different lasers are currently available and the range of these is continually
expanding. Typical lasers include the Argon ion (351, 454, 488, 514 nm), Krypton (488, 532, 630 nm), Helium neon (632
nm), Helium cadmium (325, 441 nm) and Yag (532 nm) lasers.

The Optical System

Filter
2
 PMT
 Green Light which is emitted from the cells that have
Barrier
Filters
 been illuminated in the flow chamber is directed
to an array of detectors using a complex system
Filter
1
 PMT
 Side Scatter & Blue of filters and mirrors called the optical system. A
simple configuration for a single laser instrument
is shown below.
Lens

In this example, the light source originates from a
blue laser which has passed through a focusing
FS
 Lens
 Laser
 lens and entered the flow chamber. As stated
Flow

 earlier, the physical and fluorescent characteristics
Chamber

of the cells are directly related to the character-
istics of the emitted light signals.

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY
A bar placed on the opposite side of the flow chamber blocks the laser beam. The forward light scatter (FSc) detector sits
behind the blocker bar and this detects light scatter at angles in a forward direction. Forward light scatter is an indication
of cell size.

A second focusing lens is used to direct light on to a series of filters that are used to select out light of different wave-
lengths. Long pass filters allow light above a certain wavelength to pass through and short pass filters allow light below
a certain wavelength to pass through. In the above diagram, Filter 1 selects light at wavelengths less than 500nm (blue)
which gives rise to the side scatter data (cell granularity) and Filter 2 selects light below 540nm which passes through
a green barrier filter.

The light (photons) which passes through an optical system must be converted into an electrical signal in order for the
data to be acquired and analysed. This is achieved using photodiodes (for forward scatter, i.e. size detection) and pho-
tomultiplier tubes (PMTs) for the fluorescent signals, and signals that are associated with the detection of side-scattered
light (i.e. for the measurement of cellular granularity); such components are also referred to as light detectors. Current
flow cytometers can have as many as 12 PMTs and this number will no doubt increase as the technology develops.

Computer - Data Analysis

The electronic signals that are generated by the photodiodes and PMTs are collected and processed using software
programs, some of which are dedicated to the acquisition and analysis of data from specific instruments, and others that
are free-standing and can analyse data from any instruments which have been collected in the flow cytometry standard
(.fcs) file format.

The forward and side light scatter (FSc and SSc respectively)
characteristics can be used to identify different cells in a
mixed population on the basis of cell size and granularity. In
the example to the right, neutrophil, monocytes and lympho-
cyte populations can be identified on the basis of their light
scattering characteristics alone (Data courtesy of Dr Jason
Boland, University of Sheffield).

Cellular integrity (viability), the presence of cellular components

and the expression of different proteins (amongst other
parameters) can be defined by staining cells with appropriate
fluorescently labelled molecules and antibodies. It is also
possible to investigate the interactions of cells with different
molecules by incubating them with fluorescently labelled
molecules and proteins. The fluorescent light which is
released by these dyes when they pass through the laser
excitation line is collected and directed through the optical
system.

The signals are collected by the array of detectors and processed to provide information on the fluorescent character-
istics of the cell population which is being investigated. Staining of human peripheral blood mononuclear cells with an
Alexa Fluor® 700 conjugated mAb reactive with CD8 and fluorescein isothiocyanate (FITC)-conjugated mAbs reactive
with CD56 and CD16 (identifies natural killer (NK) cells) clearly defines the two cell populations when the Alexa Fluor®
700 and FITC fluorescent characteristics of cells are plotted against each other in what is known as a ‘dot plot’ (see
below left; Data courtesy of Dr Jason Boland, University of Sheffield). In addition to being able to define whether a
particular cell is reactive with a fluorescent probe, flow cytometry can provide information on the intensity of the signal
and hence an indication of the amount of probe that has bound. As an example, stimulating CD8+ T cells via the T cell
receptor activates them and induces the expression of the early activation antigen CD69 (see below right; Data courtesy
of Hannah Cussen/Gemma Foulds (left panel) and Dr Jason Boland (right panel), University of Sheffield). It is therefore
important to consider both the proportion of cells that are exhibiting the fluorescence of interest and the intensity of that
fluorescence.

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY

The Complexities of Flow Cytometry

The range of flow cytometers which is available is expanding, and these instruments can be configured to suit the par-
ticular needs of the User. The laser and filter configurations can be customised either by the original manufacturer, or
specialised suppliers. When first developed, flow cytometers typically had a single laser and were capable of detecting
five parameters (forward light scatter, side light scatter plus 3 different fluorescent wavelengths). However, the com-
plexity of these instruments has increased considerably, with the latest models having up to 5 lasers and the capability
of measuring 20 parameters (forward light scatter, side light scatter plus 18 different fluorescent wavelengths). These
advances have made the design of experiments and analytical approaches far more difficult, as a number of issues must
be considered (see below). Although the new ‘bench top’ instrumentation which is currently available renders the physi-
cal practice of flow cytometry relatively straightforward, the acquisition of meaningful results remains a challenge and it
is essential that the investigator has a full understanding of the strengths and potential weaknesses of the technique and
the limitations of the data that are generated. This is especially the case with respect to the inclusion of appropriate controls.

The combination of fluorescent labels to be used is critical, as each one must be selected on the basis of their own
characteristics, as well as their compatibility with the other fluorescent molecules that are to be used and the instrument
which is to be used for the sample analysis.

Additional issues that must be taken into consideration include:

1. Stain Index: In its simplest terms, the Stain Index is a parameter which reflects the ability to resolve a dim positive
signal from background. In the design of multi-parameter experiments, it is better to use a fluorochrome with a low Stain
Index for measuring parameters that are expressed at high levels and a fluorochrome with a high Stain Index for
measuring parameters that are expressed at low level.

2. Spectral Overlap: Spectral Overlap occurs when the light emitted from one fluorochrome ‘leaks’ into the channel
which detects the fluorescent signal which is being emitted by another fluorochrome. The potential consequence of this
is a false positive signal. Although it is possible to eliminate this by electronically removing this signal (a process called
‘compensation), it is best avoided/minimised if possible. The concept of compensation remains one of the aspects of
flow cytometry which continues to mystify new users. Although many instruments and software packages can perform
the compensation for experimenters (assuming that the correct samples have been included in the assay), it is still im-
portant to understand the basic principles. For a two colour experiment, the easiest way to approach compensation is to
stain a sample with one fluorochrome which is known to give a signal in its ‘own’ channel and look for fluorescent signals
in the other channels that are being used. If no signal is present, then there is no spectral overlap and hence no need
to compensate. If there is a fluorescent signal in a channel which should not see one, then spectral overlap exists and
should be eliminated by subtracting the ‘inappropriate’ signal in this channel from the measurements made using the
controls of the flow cytometer. One can imagine that cross-checking spectral overlap in all possible channels becomes
progressively more challenging as the number of fluorochromes increases.

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY
3. Appropriate Controls: Flow cytometry is crucially dependent on the inclusion of appropriate controls which enable
Spectral Overlap and non-specific binding of reagents to be evaluated and considered. Incubating samples with
immunoglobulins that are labelled with the same fluorochrome as the antibodies being used, but which are known not
to react with the particular cell and species under investigation (a so called ‘isotype’ control) provides insight into any
potential non-specific binding problems. Although this is not necessarily definitive, it is a control which is widely utilised.
It is essential to fully appreciate the potential artefacts that might be present in a particular experiments and include the
necessary controls as appropriate.

4. Reagent Sourcing and Availability: The number of products and suppliers of reagents for flow cytometry is rapidly
increasing and it is becoming progressively more difficult to identify the appropriate combination of reagents for a partic-
ular flow cytometry experiment and source these from the plethora of manufacturers that are now supplying antibodies.
Furthermore, it is not always the case that the labelled antibody which is required is available and approaches that can
generate these ‘in house’ are therefore needed. In the vast majority of cases, it is not financially practical to generate a
monoclonal antibody for a particular application if this is already available from another. It is therefore important to have
the capacity to label commercially sourced antibodies in a cost effective manner whilst simultaneously overcoming the
difficulties and sample loss that are associated with traditional labelling techniques.

Lightning-Link® - Simple to Use Antibody Labelling Kits

The simplification of the antibody labelling process (most notably the elimination of column separation steps) circum-
vents many issues that have beset traditional procedures for years, i.e. loss of material, sample dilution during column
chromatography, batch-to-batch variation and difficulties in scaling up.

The Lightning-Link® process is summarized in the Figure below. The purified antibody to be labelled is transferred into
a vial of lyophilized mixture containing the label of interest. Dissolution of the vial contents activates the chemicals that
mediate the antibody labelling reaction. As there are no purification or separation steps (by products of the reaction are
completely benign), antibody recovery is close to 100%. The simplicity of the approach means that the labelling proce-
dure can be completed in less than thirty seconds. A timed demonstration of the antibody labelling process can be seen
here (Lightning-Link® video). The labeling chemistry involves the free amine groups found on lysine amino acids, and
so any antibody or protein, irrespective of isotype or species, can be labeled. Of further interest, Lightning-Link® Rapid
now comes with all the benefits of Lightning-Link® with the added advantage that the labeled antibody is ready to use
within 20 minutes from start to finish. The Lightning-Link® Rapid range now includes the DyLight® dyes from Thermo
Fisher Scientific –please see the following table for all the available dyes in the antibody labelling range:

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY
Label Lightning-Link Absorbance Emission Extinction Emission Stokes
Product Max (nm) Max (nm) Coefficient Colour Shift
(cm-1M-1)
DyLight® 350 320-0010 353 432 15,000 Blue 79
AMCA 712-0010 353 442 19,000 Blue 89
Atto 390 349-0010 390 479 24,000 Blue 89
DyLight® 405 321-0010 400 420 30,000 Blue 20
Atto 425 731-0010 436 484 45,000 Green 48
Atto 465 732-0010 453 508 75,000 Green 55
PerCP 718-0010 482 677 380,000 Red 195
PerCP/Cy5.5 763-0010 482 700 N/A Red 218
PE/Cy5 760-0010 488 670 N/A Red 182
DyLight® 488 322-0010 493 518 70,000 Green 25
Fluorescein 310-0010 494 520 73,000 Green 26
Atto 488 350-0010 501 523 90,000 Green 22
Atto 532 736-0010 532 553 115,000 Yellow 21
R-Phycoerythrin (R-PE) 703-0010 535 575 2,000,000 Orange 40
PE/Texas Red 767-0010 535 615 N/A Red 80
PE/Atto 594 768-0010 535 627 N/A Red 92
PE/Cy5.5 761-0010 535 694 N/A Red 159
PE/Cy7 762-0010 535 776 N/A Far Red 241
Rhodamine 311-0010 544 576 94,500 Orange 32
B-Phycoerythrin (B-PE) 716-0010 546 575 2,410,000 Orange 29
Cy3 340-0010 550 570 150,000 Orange 20
FluoProbes®547H 788-0010 557 574 150,000 Orange 17
DyLight® 550 323-0010 562 576 150,000 Orange 14
Atto 565 351-0010 563 592 120,000 Orange-Red 29
Cy3.5 784-0010 581 596 150,000 Orange-Red 15
FluoProbes®594 790-0010 591 616 92,000 Red 25
DyLight® 594 324-0010 593 618 80,000 Red 25
Texas Red 714-0010 595 615 80,000 Red 20
Atto 590 739-0010 600 630 120,000 Red 30
Atto 594 352-0010 601 627 120,000 Red 26
Atto 633 353-0010 629 657 130,000 Red 28
Atto 637 746-0010 630 659 120,000 Red 29
APC/Cy5.5 764-0010 633 694 N/A Red 61
APC/Cy7 765-0010 633 776 N/A Far Red 143
DyLight® 633 325-0010 638 658 170,000 Red 20
Cy5 342-0010 643 667 250,000 Red 24
Allophycocyanin (APC) 705-0010 650 670 700,000 Red 20
DyLight® 650 326-0010 652 672 250,000 Red 20
FluoProbes®647H 362-0010 653 674 250,000 Red 21
Atto 655 749-0010 663 684 125,000 Red 21
Atto 680 750-0010 670 710 125,000 Red 40
Cy5.5 782-0010 675 694 250,000 Red 19
DyLight® 680 327-0010 682 715 140,000 Red 33
Atto 700 751-0010 690 725 120,000 Red 35
Cy7 783-0010 747 776 200,000 Far Red 29
FluoProbes®682 792-0010 620-690 709 N/A Red N/A
FluoProbes®752 793-0010 650-748 772 N/A Far Red N/A
DyLight® 755 328-0010 754 776 220,000 Far Red 22
DyLight® 800 329-0010 770 794 270,000 Near Infrared 24

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A BEGINNER’S GUIDE TO FLOW CYTOMETRY
These simple antibody labelling kits are of special interest to the flow cytometry community, as there are over 40
different labels including fluorescent proteins, dyes and tandems, which span the whole light spectrum from UV to far
infrared. It is therefore now possible for the flow cytometry user to design and develop a unique set of labelled antibodies
for use in the flow cytometer using commercially sourced unlabelled antibodies. Furthermore, there are many benefits
associated with direct labelling of a primary antibody:

• Eliminates the need for the use of secondary antibodies, thus reducing the number of incubation and wash steps -
saves both time and money.

• In experiments which use several antibodies simultaneously, cross-species reactivity is not an issue as the primary
antibodies can be labelled with different dyes. With indirect detection, cross-species reactivity of secondary antibodies
is often a problem.

• Commercial sources often do not have the correct antibody conjugated to the required label. By direct labelling of the
antibody of interest yourself, this hurdle is easily overcome.

Additional information can be read in the ‘Guide to Antibody Labelling and Direct Detection.’

Summary

Flow cytometry is a powerful technique for cellular analysis which can rapidly generate complex datasets that can provide
insight into cellular status, processes and events which would be difficult, if not impossible, to achieve using other
approaches. If the reader has been inspired by this simple guide, then you are encouraged to delve deeper into the
subject by reviewing the selected resources in order to increase your knowledge base and enable the mastering of this
powerful and adaptable technique.

Bibliography and Resources

• Dean PN, Bagwell CB, Lindmo T, Murphy RF and Salzman GC. Data File Standard for Flow Cytometry.
Cytometry 1990, 11:323-332.
• Murphy RF, Chused TM:A proposal for a flow cytometric data file standard. Cytometry 1984 5:553-555.
• Robinson PJ. Mack Fulwyler in his own words. Cytometry Part A 2005, 67A:61–67
• Flow Cytometry - A Basic Introduction – Wicki Version (Michael G. Ormerod) http://flowbook-wiki.denovosoftware.com/
• Consolidated Resource for Flow Cytometry Antibodies, Instrumentation, Software & Suppliers: http://www.chromocyte.com

Innova Biosciences Ltd. Babraham Research Campus, Cambridge, UK, CB22 3AT

Phone: +44 (0)1223 496 170 Fax: +44 (0)1223 496 172
[email protected]
www.innovabiosciences.com

Lightning-Link™is a registered trademark of Innova Biosciences

Dylight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

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