the basics of flow cytometry - unibas.ch
TRANSCRIPT
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The Basics of Flow Cytometry
Janine Bögli, Biozentrum, 11. June 2020C ore
F ACS
F acility
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How to ask questions in zoom?
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The Basics of Flow Cytometry
Janine Bögli, Biozentrum, 11. June 2020C ore
F ACS
F acility
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The functions of theFACS Core Facility
Centralization of equipment and expertise
Sorter operationAdvice and
troubleshootingTrain users
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Why is Flow Cytometry important?… and what it is used for• Immunophenotyping• DNA cell cycle/tumor ploidy• Membrane potential• Ion flux• Cell viability• Intracellular protein staining• pH changes• Cell tracking and proliferation• Sorting• Redox state• Chromatin structure• Total protein• Lipids• Surface charge• Membrane fusion/runover• Enzyme activity• Oxidative metabolism• Sulfhydryl groups/glutathione• DNA synthesis• DNA degradation• Gene expression
• Analyze thousands of cells in a short time
• Statistical information obtained quickly• Flexibility of data acquisition• Ability to re-analyze
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Publications citing 'flow cytometry'
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The use of flow in research has boomed since the mid-1980s
Why we use it…
Plus manyothers!
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Overview
• What is Flow Cytometry• Fluorescence• The basics of a flow cytometer
• Fluidics• Optics• Electronics
• Data Display• How does flow cytometry data look like• Analysis and Gating
• Applications
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What is Flow Cytometry?
Cytometry
“kytos” Greek: Hollow/cell
Measurement
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The measurement canbe substrate-based…
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What is Flow Cytometry?Flow
Cytometry
“kytos” Greek: Hollow/cell
Measurement
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…or flow-based.
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What can Flow Cytometry do?
Analyze light signals to:• Enumerate particles in suspension• Evaluate 105 to 106 particles in less than 1 min• Detection of rare cell populations• Measure multiple parameters• Sort single particles for subsequent analysis
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Fluorescence
• Intrinsic fluorescence• Inheritent molecules within the cell
• Autofluorescence
• Extrinsic fluorescence• Added to the cells by investigators
• Includes dyes and fluorescent proteins
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What is Fluorescence?
• Excitation (Absorbance) with one color• Emission (Fluorescence) with a different color• Fluorophore: the part of the molecule that is fluorescent• Fluorochrome: the whole molecule (can have several
fluorophores)11
absorbedexcitationlight
emittedfluorescencelight
Jablonski DiagramFITC EmissionFITC Excitation
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Fluorochromes
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Pacific Blue FITC PE APC
Make use of spectral viewers:https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.htmlhttp://www.bdbiosciences.com/sg/research/multicolor/spectrum_viewer/index.jsphttps://www.fpbase.org/
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Fluorescent Probes
Cyan FPGreen FPYellow FPOrange FPRed FPmCherrymTomato
FITCPhycoerythrinAllophycocyaninPerCPAlexaFluor dyesPE-Cy5, PE-Cy7BV, BUV
DAPIHoechst dyesPropidium iodideAcridine OrangeTO-PRO-3DyeCycle dyesSYTOX dyes
Antibodies Fluorescent dyes Fluorescent proteins
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80’s to late 90’s
1-2 Lasers 3-4 Detectors
low dimensional data
Early 2010’s
3-5 Lasers 6-18 Detectors
higher dimensional data
Early 2000’s
1-3 Lasers 3-8 Detectors
mid dimensional data
Late 2000’s
2-4 Lasers 3-16 Detectors
mid-high dimensional data
Beyond 2015
4-9 Lasers 12-25+ Detectors
very high dimensional data
1980 20201990 2000 2010
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What is inside the Flow Cytometer?
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The Many Parts of Flow
Basic components:Fluidics Optics Electronics
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Fluidics – Schematic Overview
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Sample injectionprobe (SIT)
Interrogation point
Flow cell
waste aspirator
Interior reservoir
Sample tube
Sample Flow
Sheath Flow
Sheath filter
Sheath tank
Illumination volumeIntercept
Laser intersection point
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Fluidics – Hydrodynamic Focusing
Adapted from Purdue University Cytometry Laboratories
Sheathfluid
Injector Tip
sample
Focused Laser Beam
Forward ScatterSignal
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Fluidics – Laminar Flow • Sheath and sample fluids stream go in parallel through the
flow cell • The sample flows in the very center of the sheath• Sample and sheath fluids don’t mix
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We need to keep a smooth laminar flow!
no airno aggregates
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Fluidics – Sample Differential
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The sample pressure determines the flow rate…
Low Medium High
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Fluidics – Sample Differential
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…and thus the core diameter.
Low Medium High
Increased differential pressure will increase the sample flow rate, but will also increase the incidence of multiple cells
passing through the laser at the same time.
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Fluidics – There is a Speed Limit
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With a higher flow rate we…
• …increase coincidences• …get no single-cell analysis• …get sub-optimal data• …lose data
BAD To increase aquisitionrates you have toconcentrate your
sample
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Optics - Lasers
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• Laser light is coherent and generally of a single wavelength• Flow cytometers can have a single or up to 7 lasers (or more)
wikipedia
UV (325, 355, 375nm)
Violet (405nm)
Blue (488nm)
Green (532nm)
Yellow (561nm)
Red (633nm)
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Optics – Laser Arrangement
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Optics – Light Scattering
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• Light scattering activity depends on:• cells optical density, it’s roughness and to some extend
to its size• Scattering is dependent on the primary laser light
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Optics – Optical Arrangement
coherent light source (488nm)
flow cell
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sample stream
forward scatterdetector
Light scatters in all directions!
obscuration bar
Forward Scatter(FSC,FALS,FS)
• ~2-20° of the laserintercept
• Based on MieScatter
• Scatterproportional tothe square ofthe diameter ofthe cell
• Based on ‘sphericalparticles
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Optics – Optical Arrangement
coherent light source (488nm)
flow cell
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sample stream
forward scatterdetector
obscuration bar
Side Scatter(SSC, SS, orthogonal scatter)
• orthogonal scattering (90°)
• darkfield• complexity and
granularity
side scattercollection lens
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Optics – Optical Arrangement
coherent light source (488nm)
flow cell
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sample stream
forward scatterdetector
obscuration bar
Fluorescenceemission
• Collected with thesame lens as theSSC (15-150°)
• Intrinsic or extrinsicfluorescence
collection lens forSSC and fluorescence
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Optics – Fluorescence
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488nm
FITC
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Optics – Capture Fluorescence
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488nm
FITC
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Optics – Capture Fluorescence
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488nm
GFP
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Optics – Capture Fluorescence
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FITC /GFP
Stokes shift
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Optics – Capture Fluorescence
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PE
Stokes shift
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Optics – Capture Fluorescence
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488nm
FITC PE
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Optics – Capture Fluorescence
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488nm
FITC PE
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Optics – Optical Filters• Filters transmit light of a specific wavelength while reflecting
other wavelengths• There are three types of dichroic filters:
• Shortpass (SP) filters• Longpass (LP) filters• Bandpass (BP) filters
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Optics - Filter PropertiesLongpass filters transmit wavelengths above a cut-on wavelength
Bandpass filters transmit wavelengths in a narrow range around a specified wavelength
550LP
Emission light
> 550nmEmission light
510/20BP
500-520nm
LP
Emission light
550SP
< 550nm
Shortpass filters transmit wavelengths below a certain wavelength
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SP BP
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Optics - Dichroic Mirror
With a specific angle the filter can be used as a dichroic mirror
Transmitted Light
Light Source
Filter placed at 45o
Reflected light
Detector E Detector D
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PEFITC
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Optics –Instrument Configuration
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Know yourinstrument
configuration whenyou select for
fluorescent probes.
• Excitation spectrum: will determine whether we can use that fluorochrome
• Emission spectrum: will tell us which filter to use and whether we can combine the fluorochrome with others.
Detector E Detector D
PEFITC
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Electronics - Overview
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PMT
VGain
• photons are filtered, collected, and multiplied• The current generated is converted to a voltage pulse• The voltage pulse is digitized• The values are stored
N photons in
e-e-
e-e-
e-
Photocathode Anode
Current Out
e-e-
e-
e-e-
e-
e-e-
e-
e-e-e-
Photoelectrons
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Electronics – Light detection• Photomultipliers (PMTs) simply detect photons• Light needs to be optically filtered before• Photon energy is converted into a signal that is dependent
on:• Number of photons• Voltage applied to the PMT
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The measurement is only relative!
Controls, controls and controls!
PMT voltage setup is important
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Electronics – Signal Conversion
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Laser
Transmitted Light Signal Photons
Detector (PMT, ADP, CCD)Electrons
Volts
Time
Area AHigh H
Width W
t2
t1
t0 t1
t3
t2 t3
t0 t1 t2 t3
t0 t1 t2 t3
Quantitative MeasureCurrent
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Summary I: Channel Layout• Photon-distribution to detectors according to energy-levels
(wavelengths)• Optical elements provide separation of channels and
wavelength selection
Illumination Volume
PI, PerCP
FITC, Alexa 488, GFPPE
Flow cell
SSC
FSC
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What does a cytometer give us?
• Listmode file: correlated data file where each event is listedsequentially, parameter by parameter
• Flow cytometry standard (FCS 3.1)• Allows other software programs to recognize and analyze data• Data and header portion
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FSC SSC Time FL1 FL2 … FLnCell 1 50 20 0.001 45 2000 686
Cell 2 55 18 0.007 47 1867 600
…
Cell n 67 234 12.754 86 2134 765
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PMT Voltages• Where are my negatives?
• Signals should be withinlinear range of the detector
• Other approaches exist: • Setting the voltage based
on your cells being 2.5*rSDof the electronic noise (see Trotter, Meinelt et al, BD Biosciences Tech Bulletin)
• Quality assurance forpolychromatic flowcytometry using a suite ofcalibration beads (seeStephen P Perfetto et al, Nature Protocols (2012))
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Low voltage
Mid voltage
High voltage
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Data Display – Histogram
• The further to the right, the brighter the fluorescence• Good to display normal distributions• Show differences between samples/populations• Peak hight is a function of the CV (spread) and the number
of events
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Data Display – Dot Plot
Red
fluor
esce
nce
Green fluorescence
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Each event is plotted according to its value in the x and y dimensions
x
y
Q1 Q2Q3 Q4
FITC
APC
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Data Display – Dot Plot
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• Bivariate plots show 2 dimensions
• They provide more graphical information and more data
CD8 (fluorescence 1 red)
CD
4 (fl
uore
scen
ce2
blu
e)
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What we learn from Light Scattering
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Sid
e Sc
atte
rSSC
Forward Scatter FSCBigger
Mor
e G
ranu
lar
Live Cells
Bigger Cells or Aggregates
Dead Cells
Apoptotic Cells
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Data Display - N-Dimensional Plots
• Can rotate them• For analysis only usually• Statistics are hard to determine on these
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…and 4+ parameters?
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Data Display - N-Dimensional Plots
• We have to reduce the data• Dimensionality reduction and embedding methods• For this we define populations and gates
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New Tech - Spectral Flow Cytometry
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40 c
olor
s…
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Gates
• A region (or region of interest ROI) can be drawn on• A histogram to define boundaries and calculate percentage of
positive• A bivariate plot to define populations and calculate percentages
• Gating should be based on some scientific basis• Proper controls are essential• Follow the density of the population
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Gates• Gates can be combined and are used to isolate subsets of
data• Gates are usually hierarchical organized, like a family tree
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child grandchild
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Why Gating is useful• Eliminates false positive events and cleans up your plots• Is used to isolate a subset of cells on a plot• We can use them to select a population for further study• Allows the ability to look at parameters specific to only that
subset
54From Annual Flow Cytometry course, University Zürich
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Summary II - the Main Points• Keep a smooth laminar flow! Prevent air and aggregates.• To increase acquisition rates you have to concentrate your
sample.• The key to good results is good sample preparation.• Know your instrument configuration when selecting for
fluorescent probes.• Gating helps to define your cells of interest.• All measurements are relative, don’t forget the controls.• Include a viability dye and doublet
discrimination gating to eliminate false positive events.
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There is more…
Applications:• Major applicacions phenotyping and fluorescent proteins• Examples for special applications: RNAFlow & FRET
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Immunopheno-typing
DNA and RNA analysis
Cell deathFunctional analysis
Transduction / Transformation confirmation
Basic Use:
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Application - Immunophenotyping
• Establish the presence or level of an antigen
• Use of labeled antibodies to identify cells of interest
• Detection of cell surfacemolecules as examplecluster of differentiation
Lymphocyte T or B????
only cells fromR1
SSC
FSCwww.med4you.at T-cell marker
B-ce
llm
arke
r57
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Application - Intracellular staining
• Intracellular staining of cytokines, cytoskeleton, enzymes, transcription factors, signaling molecules
• Monitor signaling cascades
FixationPermeabilization
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Krutzik et al. 2011
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Application - DNA Analysis• DNA content of individual cells informs about their ploidy • Suitable dyes: PI, 7-AAD, DAPI
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Adapted from Beckman-Coulter
From Bitesizebio
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Application - Cell Death
Measurements of cell death:• Expression of proteins
involved in apoptosis• Activation of caspases• Changes in the
mitochondrial membrane potential
• Changes in the plasma membrane
• Cell shrinkage• Chromatin changes• DNA degradation
Normal HL60
apoptotic HL60
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Fluorescence Resonance EnergyTransfer Assays
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https://www.innovabiosciences.com/resources/applications/fret/
• protein-protein interactions with the help of fluorescently labeled proteins
• distance between the two proteins must be less than 10nm
• The emission spectrum of the donor fluorophore must overlap the absorption spectrum of the acceptor fluorophore
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Application - RNA Flow Assay
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Gene-specific oligonucleotide probe set and branched DNA technology:
• Compare RNA and protein kinetics in the same cell• Parallel analysis of microRNA targets in combination with
antibody staining• Detect target-specific RNA for which flow cytometry
antibodies are nonexistent
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Application - RNA Flow Assay
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Gene Expression
• Genes well characterized and can be cloned in frame with gene of interest
• Can be used to monitor rates of gene expression
• Commonly used as marker of transfection
• Level of intensity can be variable
• Great for cell sorting applications
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green fluorescence
yello
wflu
ores
cenc
e
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Application - Cell Sorting• Separating cells based on
properties measured in flow is also called Fluorescence-Activated Cell Sorting (FACS)
• High-speed cell sorting is based on droplet deflection
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Summary III
isac-net.orgwww.cyto.purdue.eduflowbook-wiki.denovosoftware.comlink.springer.com/content/pdf/10.1007/978-1-4939-7346-0.pdfwww.biozentrum.unibas.ch/research/groups-platforms/overview/unit/fcf/
urce
s:
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• Basic principle of every conventional flow cytometer• Quantitative measurements of single cells• High dimensionality and Multiplexing• Diverse application range
C ore
F ACS
F acility
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Controls in Flow Cytometry
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• Instrument specific controls• Fluorescence controls
• Compensation controls• Autofluorescence controls
• Experiment specific controls• Staining controls
• Non-specific binding controls• FMO controls• Secondary only controls
• Reference controls• Biological controls
Biological variabilitygreatest source of variation
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Choose well or Compensate
Violet Blue Yellow/Green RedHoechst FITC PE APC-Cy6DAPI GFP PE tamdems APCCFP Alexa 488 PI Alexa 647Pacific Blue PerCP RFP TO-ProBrilliant Violet PerCP tandems mCherry Alexa 700
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• Spectral overlap is inherent in multicolor experiments• This will have impact on experimental design and detection
sensitivity• BUT we can do something about it
• Combine fluorochromes that do not overlap• Run the right controls and compensate
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Compensation (1)
What we want…
Source: Tim Bushnell
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Compensation (2)
…and what we get
Source: Tim Bushnell
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Spillover
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100 22 …1 100 …… … 100
FITC
PEFITC
PE
How much FITC spills into others.
…
…How much fromothers spill into
FITC.
FITCdetector
PE detector
FITC signal
PE signal
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Compensation (3)
• «Compensation»• Intra-laser compensation -
more intuitive aspect• Inter-laser compensation -
less intuitive but extremely important to consider, especially with tandems
• Conceptually very simple procedure
• Introduce a linear „correction coefficient“ for all parameters that receive the spill-over
• Look at a control samples stained with a single dye, ‘single stained controls’
Source: Tim Bushnell/BD