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Flow Cytometry
1.
© Innova Biosciences
Ltd. All rights reserved Quality – Consistency – Expertise
2.
© Innova Biosciences
Ltd. All rights reserved Welcome to our 12th webinar Flow cytometry – sample preparation and experimental design Dr Andy Lane
3.
© Innova Biosciences
Ltd. All rights reserved Dr Andy Lane • Cell source and preparation • Sample treatment • Controls and maximising signal • Choosing and using antibodies
4.
© Innova Biosciences
Ltd. All rights reserved Cell source and preparation Whole blood Anti-coagulant – is your antigen sensitive to any components? e.g. binding of some CD41 antibodies is sensitive to EDTA Choice of lysis solution – ammonium chloride, or commercial solutions that fix at the same time as lysing. Occasional examples of individual antibodies being sensitive to certain formulations Most lysis solutions will work for all species, but be aware of nucleated red cells in some pathological samples and in avian species
5.
© Innova Biosciences
Ltd. All rights reserved Cell source and preparation Whole blood Interfering substances in plasma? A key example is staining of surface immunoglobulins on B lymphocytes – Ig in plasma needs to be removed as it will otherwise block antibody binding Can be achieved by washing 3x in warm isotonic buffer (J. Immunol. Meths. (1992) 151:123)
6.
© Innova Biosciences
Ltd. All rights reserved Cell source and preparation PBMC Peripheral blood mononuclear cells are prepared from whole blood using a ficoll centrifugation gradient These contain lymphocytes and monocytes, and may be used especially when culturing cells for further development or analysis. Be aware of the risk of loss of cell subsets. Cell lines Homogeneous population, although be aware that cells will be at different stages of the cell cycle. Be aware of sub-clones of well known cell lines – e.g. CD3 on Jurkat cells
7.
© Innova Biosciences
Ltd. All rights reserved Cell source and preparation Suspensions prepared from solid organs Typical examples are lymph node, spleen, liver etc Physical disaggregation or with enzymatic assistance (e.g. collagenase) Enzymes may damage cell surface antigen expression Be aware that some cells within tissues may not be released effectively. e.g. IHC staining will show many F4/80+ve macrophages within spleen, but few may be seen in flow cytometry
8.
© Innova Biosciences
Ltd. All rights reserved Location of antigen Is your target antigen at the cell surface, in the cytoplasm or in the nucleus? Is it a secreted molecule? If so, consider use of a transport inhibitor such as monensin or brefeldin A to increase cytoplasmic levels Be aware that some cell surface antigens have epitopes that are intracellular e.g. CD3, CD79a/b
9.
© Innova Biosciences
Ltd. All rights reserved Location of antigen Various options are available for permeabilising cells for intracellular staining. Two stages are required, a fixation step to stabilise the cell membrane, and a detergent based step to permeabilise cells Reference method is fixation with paraformaldehyde and permeabilisation with saponin. Be aware that in many cases detergent needs to be included throughout the staining procedure Staining of nuclear antigens may require additional steps, such as methanol Many commercial reagents available – follow instructions with antibodies wherever available
10.
© Innova Biosciences
Ltd. All rights reserved Location of antigen Combining surface and intracellular staining Consider order of adding antibodies – cell surface antigen should be stained first Some formulations may damage certain fluorochromes – e.g. a methanol modified protocol for undertaking nuclear staining may damage RPE conjugated antibodies used to stain cell surface antigens
11.
© Innova Biosciences
Ltd. All rights reserved Controls Negative and positive controls Need to differentiate between real positive signals and non-specific binding and/or auto-fluorescence Cells only control – shows baseline fluorescence of the cell population being studied Isotype control – same species of antibody, same sub-class, same dye, same concentration Be aware that an isotype control is designed to identify non-specific binding – it will not necessarily give the same result as cells alone
12.
© Innova Biosciences
Ltd. All rights reserved
13.
© Innova Biosciences
Ltd. All rights reserved Controls Negative and positive controls FMO controls (fluorescence minus one) – used in multi-colour experiments to assist with setting compensation. Include all antibodies apart from one, and ensure that there is no fluorescence overlap into that channel Positive controls – at the most basic level to confirm activity of reagents, but may also be important in confirming activation status of cells e.g. use CD25 or CD69 to confirm success of an activation protocol
14.
© Innova Biosciences
Ltd. All rights reserved Reducing background Live/dead cell discrimination – always use a dead cell dye to exclude such cells from any analysis. They tend to have high levels of auto-fluorescence, and possibly aberrant antigen expression Consider use of blocking antibodies – pre-incubate cells in serum of the same species as the antibody being used. Can be used with direct as well as indirect systems Fc block – really a more defined version of using serum as above. Best known in mouse systems using antibody 2.4G2, but several other antibodies also available with same effect Maximising signal
15.
© Innova Biosciences
Ltd. All rights reserved Reducing background Titrate your antibodies! Not all antibodies perform optimally at the same concentration – some very high affinity antibodies may be used a very high dilutions. Increasing antibody concentration above a certain point simply increases background with no increase in positive signal Maximising signal
16.
© Innova Biosciences
Ltd. All rights reserved Staining conditions Generally keep staining volumes as low as possible. If cell density is low much better to concentrate cells by centrifugation than to use a high volume for the staining step Incubation time and temperature – different opinions over staining on ice and at room temperature, and also on incubation times. Generally little difference seen by varying these within reason, but be aware that some antibodies may be more sensitive than others Remember – antibody binding is a dynamic event, you want to encourage the “on” step rather than the “off” step! Maximising signal
17.
© Innova Biosciences
Ltd. All rights reserved Selecting antibodies General principle is to use the brightest fluorochrome for the most weakly expressed antigen – “stain index” Tables of relative brightness of specific fluorochromes are available from various sources Need to understand the capabilities of the flow cytometer you are using - which lasers does it have, what filter sets are fitted/available? For multi-colour experiments choose dyes with minimal spectral overlap - various sites have excellent sources of information about fluorochrome emission spectra. Antibody choice
18.
© Innova Biosciences
Ltd. All rights reserved http://www.fluorish.com/ http://www.chroma.com/knowledge/introduction-fluorescence/fluorochrome-spectra http://www.chromocyte.com/ http://www.woodsidelogic.com/ On-line resources CytoGenie
19.
© Innova Biosciences
Ltd. All rights reserved What if the antibody conjugate I need isn’t available? Not all commercially available antibodies are available conjugated to a wide range of dyes, and even if they are your particular need may not be catered for Having a custom made conjugate prepared, either of a commercial antibody, or of your own reagent, can be expensive and time consuming Lightning-Link® conjugation kits from Innova Biosciences offer a very quick and easy-to-use solution, that requires as little as 10µg of antibody Antibody choice
20.
© Innova Biosciences
Ltd. All rights reserved What is Lightning-Link® technology? The worlds fastest, easiest to use and most efficient conjugation technology! • Only 30 seconds hands-on time! • Over 50 labels available including: Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin Lightning-Link® Antibodies – Proteins – Peptides Fast – Easy-to-use – Reliable
21.
© Innova Biosciences
Ltd. All rights reserved What is Lightning-Link® technology? • 100% antibody recovery • Fully scalable from R&D (10µg<) to Production / Manufacture (>1g) • Virtually eliminates batch to batch variability • Coupling of the label to antibodies, proteins or other biomolecules • Covalent conjugation ensures long term stability • Available as traditional Lightning-Link® (3 hour incubation) or new Lightning-Link® RAPID (15 minute incubation) Lightning-Link®
22.
© Innova Biosciences
Ltd. All rights reserved Lightning-Link® 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 A selection of flow cytometry dyes available in Lightning-Link® kits Grouped by the most commonly used excitation lasers
23.
© Innova Biosciences
Ltd. All rights reserved Conjugation considerations You need to know some things about your reagent. Lightning-Link conjugations are really simple but you need protein in the right format to work effectively. 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 Lightning Link kits are optimised for antibody labelling, but can easily be adjusted to label other proteins. If you know the size of your protein you can calculate how to use Lightning-Link for your application
24.
© Innova Biosciences
Ltd. All rights reserved Anti-human CD4 (RPA-T4) was purchased in pre-conjugated form and in unconjugated form for conjugation using Lightning-Link® kits CD4 FITC Red curve = Lightning-Link® Blue curve = Leading Ab supplier Lightning-Link® Comparative Data
25.
© Innova Biosciences
Ltd. All rights reserved RPE – blue = Ab supplier red = LL Anti-human CD54 (HCD54) was purchased in pre-conjugated form and in unconjugated form for conjugation using Lightning-Link® kits Leading Ab supplier Lightning-Link® Overlay FITC – blue = Ab supplier red = LL A mouse monoclonal antibody, clone HCD54, specific for human CD54 was purchased from a commercial source in both unconjugated and RPE or FITC conjugated formats. The unconjugated antibody was linked to RPE or FITC using a Lightning-Link® kit, and the two conjugates were compared in flow cytometry staining human peripheral blood lymphocytes, where the blue curve shows unstained cells. Lightning- Link® conjugated antibody is shown in the middle, and commercially conjugated antibody on the left hand side. The right hand histograms show an overlay of the positively stained cells. Lightning-Link® Comparative Data
26.
© Innova Biosciences
Ltd. All rights reserved 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
27.
© Innova Biosciences
Ltd. All rights reserved 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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