Flow Cytometry

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Quality – Consistency – Expertise

Welcome to our 12th webinar
Flow cytometry – sample preparation and
experimental design
Dr Andy Lane

Dr Andy Lane
• Cell source and preparation
• Sample treatment
• Controls and maximising signal
• Choosing and using antibodies

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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®

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

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

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

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

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

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