This document provides an overview of flow cytometry including: - An introduction to flow cytometry techniques and applications from multiple speakers - Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes - Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis - A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments

1. in association with

2. Welcome to our first webinar
A beginner's guide to flow cytometry and all you
ever need to know about preparing fluorescent
conjugated antibodies.

3. Prof Graham Pockley
• What is flow cytometry?
• Instruments and components
necessary for the technique
• Single and multiple colour cytometry
• Examples and applications
• Cell sorting
Speakers
Prof Pockley is Associate Director of the John van
Geest Cancer Research Centre in Nottingham
and is the founder of Chromocyte

4. Speakers
Dr Andy Lane
• Key role of antibodies for multi-colour
flow cytometry
• Antibody conjugation methods
Dr Lane has recently joined Innova Biosciences,
where he is well positioned to utilise his antibody
conjugation and flow cytometry experience in
combination with Innova’s ground-breaking rapid
conjugation technology.

5. Prof Graham Pockley
On the defining aspects, techniques and applications of
flow cytometry

6. Wikipedia
‘Flow cytometry is a technique for counting, examining, and
sorting microscopic particles suspended in a stream of fluid. It
allows simultaneous multiparametric analysis of the physical
and/or chemical characteristics of single cells flowing through
an optical and/or electronic detection apparatus’

7. • 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
Some applications of flow cytometry
Plus many others!

8. Brief History of Flow Cytometry
• The first fluorescence-based flow cytometry device was
developed in 1968 by Wolfgang Göhde (University of Münster,
Germany) and such instruments were first commercialized by
Partec in Göttingen in 1968/69
see www.coulterflow.com/bciflow/history.php
Wolfgang Göhde
• The original name of the flow cytometry technology was pulse cytophotometry
(Impulszytophotometrie in German, ICP) and this was changed to flow cytometry at
the Conference of the American Engineering Foundation in Pensacola, Florida in
1978

9. Brief History of Flow Cytometry (cont)
• The ability to measure multiple parameters (volume, light scatter, fluorescence)
using a single instrument was developed by Paul Mullaney, and the capacity to
measure side scatter was developed by Gary Salzman.
• Mack Fulwyler working in Marvin van Dilla's laboratory at the Los Alamos National
Laboratories, USA developed the sorter in 1965 (see Robinson JP, 2005).
• Leonard Herzenberg (Stanford University, USA) coined the term, Fluorescence
Activated Cell Sorter (FACS) in the mid-1970s.
see www.coulterflow.com/bciflow/history.php

10. Early instruments
seewww.coulterflow.com/bciflow/history.php
ICP 11 (1969) Distributed by Phywe, Göttingen The first commercial flow
cytometer PDP 11 computer
Epics II 1975, Designed by Mack Fulwyler
and Jim Corell Delivered to NCI/NIH
TPS 1974 - 1979, Designed by Bob Auer

11. Modern Instruments
CyAn™ ADP Analyzer
Attune® Acoustic Focusing Cytometer
MoFlo® Astrios™

12. CytoSub CytoBuoy
Flow Cytometry is not restricted to the laboratory!

13. Flow Cytometry is not restricted to the laboratory!
HIV/AIDS immune status monitoring in remote and rural areas

14. 3 core systems – fluidics, optics, electronics

15. Fluidics - The Flow Cell

16. Optics and Electronics – generation of light and its collection in
simple terms
Electronics convert light signal to something
that can be visualised by software
Typical lasers: 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.
Photodiode

17. Key parameters measured are scatter and
fluorescence

18. Scatter parameters & how they are measured
Data courtesy of Dr Jason Boland, University
of Sheffield

19. Wikipedia
Fluorescence is the emission of light by a substance that has absorbed
light or other electromagnetic radiation

20. Fluorescent Excitation and Emission
Wavelength (nm)
Relativeintensity
Stokes shift
Absorption
Emission

21. Typical
Fluorochromes
Name Laser Line Peak Emission (nm)
DyLight® 405 405 420
Atto425 405 484
DyLight® 488 488 518
Fluorescein 488 520
Atto488 488 523
Atto532 488 553
PerCP 488 677
PerCP-Cy5.5 488 695
R-Phycoerythrin (RPE) 488/561 578
PE-Texas Red 488/561 615
PE/Atto594 488/561 627
PE/Cy5 488/561 667
PE/Cy5.5 488/561 695
PE/Cy7 488/561 785
DyLight® 550 561 576
Atto565 561 592
Cyanine Dye 3.5 (Cy3.5) 561 596
Atto633 633/635 657
DyLight® 633 633/635 658
Atto637 633/635 659
Cyanine Dye 5 (Cy5) 633/635 667
Allophycocyanin (APC) 633/635 670
DyLight® 650 633/635 672
FluoProbes647H 633/635 675
Atto655 633/635 684
APC/Cy5.5 633/635 694
APC/Cy7 633/635 776

22. Ways in which antibodies bind to cells
Antigen specific:
Fab to epitope
Specific, but antigen non-specific:
e.g. Fc to Fc receptor
Non-specific:
Binding is low affinity and not saturable

23. Data courtesy of Hannah Cussen/Gemma Foulds (left panel) and Dr
Jason Boland (right panel), University of Sheffield
Data Analysis: Dot Plots vs. Histograms

24. Data Analysis

25. Two colour dot-plots

26. Data Outputs
• Proportion of cells positive for a given antigen (expressed
as a percentage)
• The fluorescent intensity
- indicative of the intensity of expression

27. Dead cells can be a problem
• They bind antibodies non-specifically
• They ‘masquerade’ as specific subsets
• They cause data misinterpretation
Always use a viability / dead cell stain!

28. 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.
Although it is possible to eliminate this by
electronically removing this signal (a process
termed ‘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.
FL-1 FL-2
A
B

29. • Some fluorochromes are ‘brighter’ than others
• In its simplest terms, the Stain Index is a parameter which reflects
the ability to resolve a dim positive signal from background
• 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
• Minimise spillover / Spectral overlap
Stain Index
FL-1 FL-2
A
B

30. Cell Sorting: concepts and applications
From: http://en.wikipedia.org/wiki/Flow_cytometry
Sabban, Sari (2011) Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high- affinity
FcεRI receptor (PhD thesis), The University of Sheffield
The acronym FACS is trademarked and
owned by Becton, Dickinson and
Company

31. Dr Andy Lane
On the role of antibodies as tools for harnessing the
technology of flow cytometry

32. Seventeen-colour flow cytometry:
unravelling the immune system
Stephen P. Perfetto, Pratip K. Chattopadhyay and Mario
Roederer
Nature Reviews Immunology 2004
Evaluation of a 12-color flow cytometry panel
to study lymphocyte, monocyte, and dendritic
cell subsets in humans.
Autissier et al
Cytometry A 2010

33. 33
Some antibodies are not
commercially available
in conjugated form
Multi-colour experiments require a wide range
of conjugated antibodies

34. 34
Some antibodies are not
commercially available
in conjugated form
Secondary antibodies conjugated
to other dyes may appear
to be an option

35. 35
Some antibodies are not
commercially available
in conjugated form
…..but those secondary antibodies will bind to the
other primary antibodies as well

36. in association with
© Innova Biosciences ltd. 2012. All rights reserved
Traditional conjugation
37
• Experienced staff with immunochemistry knowledge
• Unconjugated antibody – purified, 3-5mg
• Separation columns and possibly fraction collectors etc
• Yield issues
• Time
• Cost

37. Features of
Lightning-Link®
• Lightning-Link ® - the world’s easiest antibody labeling kits
• Simple, one step process
• Only 30 seconds hands-on
• Reproducible
• Scalable µg to mg
• 100% recovery
Just add primary antibody !
39

38. 40

39. Name Laser Line Peak Emission (nm)
DyLight® 405 405 420
Atto425 405 484
DyLight® 488 488 518
Fluorescein 488 520
Atto488 488 523
Atto532 488 553
PerCP 488 677
PerCP-Cy5.5 488 695
R-Phycoerythrin (RPE) 488/561 578
PE-Texas Red 488/561 615
PE/Atto594 488/561 627
PE/Cy5 488/561 667
PE/Cy5.5 488/561 695
PE/Cy7 488/561 785
DyLight® 550 561 576
Atto565 561 592
Cyanine Dye 3.5 (Cy3.5) 561 596
Atto633 633/635 657
DyLight® 633 633/635 658
Atto637 633/635 659
Cyanine Dye 5 (Cy5) 633/635 667
Allophycocyanin (APC) 633/635 670
DyLight® 650 633/635 672
FluoProbes647H 633/635 675
Atto655 633/635 684
APC/Cy5.5 633/635 694
APC/Cy7 633/635 776
41
A selection of flow cytometry dyes
available in Lightning-Link® kits
Grouped by the most commonly
used excitation lasers

40. How do you choose
your dye?
• What laser (s) do you have available?
• Level of antigen expression – use brighter dyes
for weakly expressed antigens
• What other dyes are being used – will they
overlap, do you need to compensate or
change filters
42

41. Fluorescence overlap
Emission spectra may overlap – in this example FITC and RPE are shown
This may be reduced by the use of filters, but overlap may remain (see A and B)
FL-1 FL-2
A
B

42. Compensation in practice
Fluorescence overlap can
be removed by adjusting
compensation settings
on the flow cytometer, or
more commonly nowadays
within software during
analysis
Uncompensated Undercompensated
Correctly compensated Overcompensated

43. © Innova Biosciences ltd. 2012. All rights reserved© Innova Biosciences ltd. 2012. All rights reserved
in association with
Filters
• Filters within flow cytometers are rarely changed by the user,
but this is possible in some instruments, and may be useful to
get the best performance from a particular dye
• Common nomenclature includes bandpass filters (eg.
530/30BP, which detects light in the range 515-545nm) and
longpass filters (e.g. 650LP) which only let light longer than
650nm pass through
• If you had a 650LP filter in place and want to detect a dye
emitting at 640nm an alternative would be needed
45

44. in association with
© Innova Biosciences ltd. 2012. All rights reserved
Conjugation
considerations
Antibody doesn’t meet
these criteria?
You need to know some things about your antibody.
Lightning Link conjugations are really simple
but you need antibody in the right format to work effectively.
Commercially available antibodies come in many forms,
and you may need to check with the supplier about some details.
Concentration – 1mg/ml or higher is preferred
Purity – ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation – most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM
Use a purification kit to purify, concentrate
and/or change the buffer of your antibody

45. in association with
© Innova Biosciences ltd. 2012. All rights reserved47

46. Using your
new conjugates
• Use exactly as normal in terms of staining technique
• Titrate – possibly extensively!
• Storage – at 40C in concentrated form is always best.
• A preservative (e.g. 0.05% w/v sodium azide) may be useful, and if
stored diluted a carrier protein would be advised (e.g. 1% w/v BSA)
• Some conjugates may be safely frozen, but others should not be.
Never freeze RPE, APC or their tandem forms!
• Keep conjugates away from light – tandem dyes are especially
sensitive48

47. will be attending the following conference
It’s free to attend and we’d love it if you came by the booth!

48. Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

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