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

Transcription

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 Speakers Prof Graham Pockley What is flow cytometry? Instruments and components necessary for the technique Single and multiple colour cytometry Examples and applications Cell sorting 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 Some applications of flow cytometry DNA/Cell Cycle analysis Cell proliferation Multicolor phenotyping (cell surface) Monocyte oxidative burst Neutrophil oxidative burst Microbiological analysis Cellular and antibody or complementmediated cytotoxicity Cell viability Intracellular ionic (e.g. Ca 2+ ) fluxes Multicolor phenotyping (intracellular) Monocyte phagocytosis Neutrophil phagocytosis Cell trafficking Sorting on the basis of morphology (FSC or SSc) and/or fluorescent characteristics 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 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 see

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

10 Early instruments ICP 11 (1969) Distributed by Phywe, Göttingen The first commercial flow cytometer PDP 11 computer see TPS , Designed by Bob Auer Epics II 1975, Designed by Mack Fulwyler and Jim Corell Delivered to NCI/NIH

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

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

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 Photodiode 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.

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 Relative intensity Fluorescent Excitation and Emission Stokes shift Absorption Emission Wavelength (nm)

21 Typical Fluorochromes Name Laser Line Peak Emission (nm) DyLight Atto DyLight Fluorescein Atto Atto PerCP PerCP-Cy R-Phycoerythrin (RPE) 488/ PE-Texas Red 488/ PE/Atto / PE/Cy5 488/ PE/Cy / PE/Cy7 488/ DyLight Atto Cyanine Dye 3.5 (Cy3.5) Atto / DyLight / Atto / Cyanine Dye 5 (Cy5) 633/ Allophycocyanin (APC) 633/ DyLight / FluoProbes647H 633/ Atto / APC/Cy / APC/Cy7 633/

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

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. FL-1 FL-2 Although it is possible to eliminate this by electronically removing this signal (a process termed compensation), it is best avoided/minimised if possible. B A The concept of compensation remains one of the aspects of flow cytometry which continues to mystify new users.

29 Stain Index 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 FL-1 FL-2 B A

30 Cell Sorting: concepts and applications From: 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 Multi-colour experiments require a wide range of conjugated antibodies 33 Some antibodies are not commercially available in conjugated form

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

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

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

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) 41 DyLight Atto DyLight Fluorescein Atto Atto PerCP PerCP-Cy R-Phycoerythrin (RPE) 488/ PE-Texas Red 488/ PE/Atto / PE/Cy5 488/ PE/Cy / PE/Cy7 488/ DyLight Atto Cyanine Dye 3.5 (Cy3.5) Atto / DyLight / Atto / Cyanine Dye 5 (Cy5) 633/ Allophycocyanin (APC) 633/ DyLight / FluoProbes647H 633/ Atto / APC/Cy / APC/Cy7 633/ 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 42 What other dyes are being used will they overlap, do you need to compensate or change filters

41 Fluorescence overlap FL-1 FL-2 B A 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)

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 Filters in association with 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 nm) 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 Innova Biosciences ltd All rights reserved

44 Conjugation considerations in association with 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. Purity ensure other proteins have been removed, and also make sure they haven t been put back again afterwards! Concentration 1mg/ml or higher is preferred 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 Antibody doesn t meet these criteria? Use a purification kit to purify, concentrate and/or change the buffer of your antibody Innova Biosciences ltd All rights reserved

45 in association with 47 Innova Biosciences ltd All rights reserved

46 Using your new conjugates Use exactly as normal in terms of staining technique Titrate possibly extensively! Storage at 4 0 C 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 48 sensitive

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 Please keep an eye out for our future webinars and other exciting news on our website and social media channels: YouTube:

49 Innova Biosciences Ltd. Babraham Research Campus, Cambridge, UK, CB22 3AT Lightning-Link is a registered trademark of Innova Biosciences DyLight is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries