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in association with
Welcome to our first webinar
A beginner's guide to flow cytometry and all you
ever need to know about preparing fluorescen...
Prof Graham Pockley
• What is flow cytometry?
• Instruments and components
necessary for the technique
• Single and multip...
Speakers
Dr Andy Lane
• Key role of antibodies for multi-colour
flow cytometry
• Antibody conjugation methods
Dr Lane has ...
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 ...
• DNA/Cell Cycle analysis • Cell viability
• Cell proliferation • Intracellular ionic (e.g. Ca2+) fluxes
• Multicolor phen...
Brief History of Flow Cytometry
• The first fluorescence-based flow cytometry device was
developed in 1968 by Wolfgang Göh...
Brief History of Flow Cytometry (cont)
• The ability to measure multiple parameters (volume, light scatter, fluorescence)
...
Early instruments
seewww.coulterflow.com/bciflow/history.php
ICP 11 (1969) Distributed by Phywe, Göttingen The first comme...
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 someth...
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)
Relativeintensity
Stokes shift
Absorption
Emission
Typical
Fluorochromes
Name Laser Line Peak Emission (nm)
DyLight® 405 405 420
Atto425 405 484
DyLight® 488 488 518
Fluores...
Ways in which antibodies bind to cells
Antigen specific:
Fab to epitope
Specific, but antigen non-specific:
e.g. Fc to Fc ...
Data courtesy of Hannah Cussen/Gemma Foulds (left panel) and Dr
Jason Boland (right panel), University of Sheffield
Data A...
Data Analysis
Two colour dot-plots
Data Outputs
• Proportion of cells positive for a given antigen (expressed
as a percentage)
• The fluorescent intensity
- ...
Dead cells can be a problem
• They bind antibodies non-specifically
• They ‘masquerade’ as specific subsets
• They cause d...
Spectral Overlap
Spectral Overlap occurs when the light emitted
from one fluorochrome ‘leaks’ into the channel
which detec...
• Some fluorochromes are ‘brighter’ than others
• In its simplest terms, the Stain Index is a parameter which reflects
the...
Cell Sorting: concepts and applications
From: http://en.wikipedia.org/wiki/Flow_cytometry
Sabban, Sari (2011) Development ...
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
Roed...
33
Some antibodies are not
commercially available
in conjugated form
Multi-colour experiments require a wide range
of conj...
34
Some antibodies are not
commercially available
in conjugated form
Secondary antibodies conjugated
to other dyes may app...
35
Some antibodies are not
commercially available
in conjugated form
…..but those secondary antibodies will bind to the
ot...
in association with
© Innova Biosciences ltd. 2012. All rights reserved
Traditional conjugation
37
• Experienced staff wit...
Features of
Lightning-Link®
• Lightning-Link ® - the world’s easiest antibody labeling kits
• Simple, one step process
• O...
40
Name Laser Line Peak Emission (nm)
DyLight® 405 405 420
Atto425 405 484
DyLight® 488 488 518
Fluorescein 488 520
Atto488 4...
How do you choose
your dye?
• What laser (s) do you have available?
• Level of antigen expression – use brighter dyes
for ...
Fluorescence overlap
Emission spectra may overlap – in this example FITC and RPE are shown
This may be reduced by the use ...
Compensation in practice
Fluorescence overlap can
be removed by adjusting
compensation settings
on the flow cytometer, or
...
© Innova Biosciences ltd. 2012. All rights reserved© Innova Biosciences ltd. 2012. All rights reserved
in association with...
in association with
© Innova Biosciences ltd. 2012. All rights reserved
Conjugation
considerations
Antibody doesn’t meet
t...
in association with
© Innova Biosciences ltd. 2012. All rights reserved47
Using your
new conjugates
• Use exactly as normal in terms of staining technique
• Titrate – possibly extensively!
• Stora...
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...
Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a r...
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A Beginner's Guide to Flow Cytometry

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A Beginner's Guide to Flow Cytometry

  1. 1. in association with
  2. 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. 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. 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. 5. Prof Graham Pockley On the defining aspects, techniques and applications of flow cytometry
  6. 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. 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. 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. 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. 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. 11. Modern Instruments CyAn™ ADP Analyzer Attune® Acoustic Focusing Cytometer MoFlo® Astrios™
  12. 12. CytoSub CytoBuoy Flow Cytometry is not restricted to the laboratory!
  13. 13. Flow Cytometry is not restricted to the laboratory! HIV/AIDS immune status monitoring in remote and rural areas
  14. 14. 3 core systems – fluidics, optics, electronics
  15. 15. Fluidics - The Flow Cell
  16. 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. 17. Key parameters measured are scatter and fluorescence
  18. 18. Scatter parameters & how they are measured Data courtesy of Dr Jason Boland, University of Sheffield
  19. 19. Wikipedia Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation
  20. 20. Fluorescent Excitation and Emission Wavelength (nm) Relativeintensity Stokes shift Absorption Emission
  21. 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. 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. 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. 24. Data Analysis
  25. 25. Two colour dot-plots
  26. 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. 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. 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. 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. 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. 31. Dr Andy Lane On the role of antibodies as tools for harnessing the technology of flow cytometry
  32. 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. 33 Some antibodies are not commercially available in conjugated form Multi-colour experiments require a wide range of conjugated antibodies
  34. 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. 35 Some antibodies are not commercially available in conjugated form …..but those secondary antibodies will bind to the other primary antibodies as well
  36. 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. 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. 38. 40
  39. 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. 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. 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. 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. 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. 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. 45. in association with © Innova Biosciences ltd. 2012. All rights reserved47
  46. 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. 47. will be attending the following conference It’s free to attend and we’d love it if you came by the booth!
  48. 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. 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

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