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
This slide show forms part of the Introduction to Flow Cytometry seminar help by The Garvan MLC Flow Cytometry Facility. The Garvan MLC Flow Cytometry Facility is part of the Garvan Institute of Medical Research and is located in Sydney NSW.
This slide show forms part of the Introduction to Flow Cytometry seminar help by The Garvan MLC Flow Cytometry Facility. The Garvan MLC Flow Cytometry Facility is part of the Garvan Institute of Medical Research and is located in Sydney NSW.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
It is a laser based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
It is a laser based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
ELISA is a well know term that is an abbreviation of Enzyme Linked Immunosorbent Assay. This microplate based technique relies on the use of an antibody that has been linked to an enzyme. In the presence of an appropriate substrate, enzymatic activity produces a color change as the ELISA readout, which can be measured and provides information about the presence and quantity of the target antigen in the sample material.
Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a principle tool in analytical chemistry, biochemistry, and molecular biology. Polyacrylamide gel electrophoresis (PAGE) can be used to analyze the size, amount, purity, and isoelectric point of polypeptides and proteins. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis (SDS PAGE) is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this technique.
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins. Ab-Oligo conjugates have since played a significant role in enhancing an extensive range of biological techniques that include immunological and proteomic research, biomarker discovery, clinical diagnostics – including point-of-care, as well as other novel techniques. Antibodies can be readily conjugated to oligonucleotides via their amino acid residues, making them suitable for most in vitro applications, as they possess several functional groups.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
GELFrEE® 8100 Fractionation System Tech NoteExpedeon
Successful sample preparation is a key step during any analytical
procedure and begins with a defined experimental design. Important steps in sample preparation include proteolytic digestion of proteins into peptide fragments, and peptide fractionation. This is especially important prior to applications such as mass spectrometry (MS).
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins.
Proteomics of small proteins from plant tissuesExpedeon
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NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Circular dichroism spectroscopy is an analytical technique used to estimate the secondary and tertiary structure of proteins. This technique can be used to confirm whether structure has been retained during protein processing, but is frequently adversely affected by additives such as solubility enhancers and detergents.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Protein processing and production is often hampered by the formation of aggregates that restrict and complicate
the handling of proteins, antibodies and enzymes. NVoy is designed to minimise the sequential losses in consecutive
protein processing steps which would otherwise dramatically reduce the overall protein yield.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Top down proteomics of soluble and integral membrane proteinsExpedeon
Mitochondria provide important cellular functions including
oxidative phosphorylation, fatty acid biosynthesis, and acting as
gatekeepers to apoptosis.
GELFrEE1 affords rapid mass-based protein separation over a range 10-150 kDa. Here, we demonstrate a multiplexed design enabling increased loading capacity and throughput. We
demonstrate comprehensive analysis of the yeast proteome using GELFrEE coupled to LC-MS/MS analysis.
Identification and characterization of intact proteins in complex mixturesExpedeon
The ability to fully characterize proteins in their intact forms allows thorough biological investigation of the functional importance of changes such as post-translational modifications, protein isoforms/sequence variations, and protease cleavages.
Improved coverage of the proteome using gel eluted liquidExpedeon
It has long been understood that sample fractionation is critically important to generating quality, comprehensive proteomics data. In spite of the continual improvements in speed and sensitivity of mass spectrometers, these instruments are still unable to adequately overcome the enormous challenge
of most biological samples without multiple dimensions of separation prior to mass analysis.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Characterization of intact antibodies by pre-fractionation using gel electrop...Expedeon
Antibodies represent an important class of proteins due to their central role in the immune response. Moreover, there is an increasing interest in the use of recombinant antibodies as novel drug therapies.
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Execution from the test manager
Orchestrator execution result
Defect reporting
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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
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
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
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
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
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
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