a beginner's guide to flow cytometry
TRANSCRIPT
Welcome to our first webinar
A beginner's guide to flow cytometry and all you ever need to know about preparing fluorescent
conjugated antibodies.
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
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.
Prof Graham Pockley
On the defining aspects, techniques and applications of flow cytometry
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’
• 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!
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
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
Early instruments
see ww
w.co
ulterflo
w.co
m/b
ciflow
/histo
ry.ph
p
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
Modern Instruments
CyAn™ ADP Analyzer Attune® Acoustic Focusing Cytometer
MoFlo® Astrios™
CytoSub CytoBuoy
Flow Cytometry is not restricted to the laboratory!
Flow Cytometry is not restricted to the laboratory!
HIV/AIDS immune status monitoring in remote and rural areas
3 core systems – fluidics, optics, electronics
Fluidics - The Flow Cell
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
Key parameters measured are scatter and fluorescence
Scatter parameters & how they are measured
Data courtesy of Dr Jason Boland, University of Sheffield
Wikipedia
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation
Fluorescent Excitation and Emission
Wavelength (nm)
Rel
ativ
e in
ten
sity
Stokes shift
Absorption
Emission
Typ
ical
Fl
uo
roch
rom
es
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
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
Data courtesy of Hannah Cussen/Gemma Foulds (left panel) and Dr Jason Boland (right panel), University of Sheffield
Data Analysis: Dot Plots vs. Histograms
Data Analysis
Two colour dot-plots
Data Outputs
• Proportion of cells positive for a given antigen (expressed as a percentage) • The fluorescent intensity - indicative of the intensity of expression
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!
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
• 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
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
Dr Andy Lane
On the role of antibodies as tools for harnessing the technology of flow cytometry
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
Some antibodies are not commercially available in conjugated form
Multi-colour experiments require a wide range of conjugated antibodies
34
Some antibodies are not commercially available in conjugated form
Secondary antibodies conjugated to other dyes may appear to be an option
35
Some antibodies are not commercially available in conjugated form
…..but those secondary antibodies will bind to the other primary antibodies as well
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
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
40
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
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
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
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
© 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
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
in association with
© Innova Biosciences ltd. 2012. All rights reserved 47
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 sensitive 48
will be attending the following conference
It’s free to attend and we’d love it if you came by the booth!
Contact
If you would like any more information, please contact us at [email protected]
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
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