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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

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ciflow

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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

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Some antibodies are not commercially available in conjugated form

Multi-colour experiments require a wide range of conjugated antibodies

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Some antibodies are not commercially available in conjugated form

Secondary antibodies conjugated to other dyes may appear to be an option

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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

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• 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 !

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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

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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

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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

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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

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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 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

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