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Suggested Day 1
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An Introduction to Flow
CytometryBy
Max Parker-Shames,
Phoebe Parker-Shames,
Marie Keil, Natalie Edson
This Publication was made possible by Grant #024094 from NIAID.
Its contents are solely the responsibility of the authors and do not necessarily represent
the official views of the NIH.
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Abdcerotec.com
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What is Flow Cytometry?
Lets break it down:
Cyto = Cell Metry = Measure
So Cytometry= measure cells
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Basic Flow Cytometers
Flow Cytometers aremachines that measuremultiple aspects of
single cells
The cells areinterrogated (examined)by lasers
They are interrogatedas they flow by in astream of fluid.
http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/
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What are Flow Cytometers
used for? Medical Research (especially in cancer
research and immune functions)
Medical Diagnoses (For detecting ormonitoring diseases like HIV/AIDS or formonitoring transplants)
Marine Biology Protein Engineering
Pathology
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How do we measure things?
Human Senses
Sight
Sound
Smell
Touch
Taste
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What does flow cytometry
measure about cells? Size
Shape (Granularity)
Makeup (Surface proteins)
Density
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HOW?
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Flow Cytometers are made of
three basic parts Optics (where the cells are analyzed by
the lasers)
Electronics (where light is translatedinto electrons and the cells are sorted)
Computer Analysis (where the data is
analyzed using software like Flowjo)
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Lets look briefly at how they
work
http://www.unsolvedmysteries.ore
gonstate.edu/flow_06
http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_067/31/2019 Physics Presentation(1)
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Cytometers work by examining
fluorescent markers They are like dyes, and are added to samples
before they are run through the cytometer.
Most are proteins. They bind to cells and give off light whenstimulated by a laser.
Some common ones used are FITC,
TRITC, NHS, and PE. We graph the relative amounts of these
markers to distinguish cells.
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Luciferin
(derived from fireflies)
Fluorescein Isothiocyanate
(FITC)
http://en.wikipedia.org/
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Amount of Blue Markers
AmountofYe
llow
Markers
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BUT
In real-world examples, the graphs willlook a lot more complicated.
Most cytometry samples containthousands of cells, not just four.
As long as you remember that they
follow the same principal, youll do fine.
Lets see an example:
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Forward Scatter (ForSc)
SideScatter(
OrthSc)
Each one of these dotsrepresents a single cell!
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Suggested Day 2
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Cell Sorting
The Fluidic System
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Fluidic System
In order to do analyses in FlowCytometers, the machine needsto analyze cells individually
To accomplish this, cells are run
through the machine in fast-moving stream of fluid (hencethe name flowcytometry)
This process is calledHydrodynamic Focusing
http://www.bdbiosciences.com
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The sample fluid stream is directed through the laser by thesheath fluid
The sample fluid is always a higher pressure than the sheath
fluid The relative pressure in the sample fluid controls the velocity of
the stream as it flows through the laser beam, or interrogationpoint
Low samplepressure, lowaverage cellcount. Thisgives greater
accuracy, buttakes longer
High samplepressure, highaverage cellcount. Thisgives lessaccuracy, butis much faster
http://www.bdbiosciences.com
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A Review of Properties of
LightWavelength, color, etc
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Wavelength,
Sinusoidal wave
v = f
Can be sound, light,or water waves
http://en.wikipedia.org/
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Colors
488 nm wavelength is the most commonly used type of laser inFlow Cytometers
http://antonine-education.co.uk/
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Lasers
Light Amplification byStimulated Emission ofRadiation
Lasers are almostalways a coherent lightsource (they haveuniform wavelength)
Usually, multiple lasersare used in FlowCytometers to analyzecells
http://plaza.ufl.edu/
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The Optics System
Excitation
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Excitation
Multiple Lasers are used inFlow Cytometers to excitethe cells
Fluorescent markers absorb
and reemit differentwavelengths
Different types of cellsscatter different colored light,this helps identify what kind
of cells they are
http://www.bdbiosciences.com
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Lenses collect emitted light and filters route
specific wavelengths into detectors It is important to keep this as exact as
possible to avoid overlapping colors
Light Sorting
http://www.bdbiosciences.com
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Electrostatic Flow
Sorting
After the cells are
interrogated by thelaser, vibrationsseparate the samplestream into dropletscontaining either oneor zero cells, called thebreak-off point
http://cyto.perdue.edu/
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At the point at which thestream breaks into droplets,it passes through anelectrically charged ringwhich charge cells based onthe results detected by thecytometers laser and
detector system
The cells then pass bycharged plates which sortthe cells based on thecharge that they have been
given
http://www.bdbiosciences.com
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Suggested Day 3
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Lets review what weve
learned and look at how theprocess works
T-Shirt sorting activity!
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Electronics System
So what happens after the cells
are interrogated by the laser andsorted by type?
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What does the electronics
system do? Converts the reflected
light into electrons
Converts analogsignals to digital
Performs
compensation Transfers data to the
computer
http://www.bdbiosciences.com
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The electronics system takes
the data from collection tocomputer
http://www.bdbiosciences.com
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Photomultipliers and
Photodiodes These are thetwo types ofdetectors thatconvertphotons intoelectricalsignals
They control
sensitivity byadjustingvoltage
http://www.bdbiosciences.com
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Compensation
There is some overlap between the colors emitted by differentfluorescent markers, therefore mathematical compensation isused to reduce overlapping results
A veryimportantfunction of theelectronics
system is toperformcompensation
http://www.bdbiosciences.com
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Suggested Day 4
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Heres a video overview of
Flow Cytometryhttp://www.youtube.com/watch?v=
nAfL4FXju1s
http://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1shttp://www.youtube.com/watch?v=nAfL4FXju1s7/31/2019 Physics Presentation(1)
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Analyzing Flow Cytometry
Data
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Where does the data come
from?
Cell samples are collected for use in the flow cytometer.
Cells come from non-solid sources, like blood.
Sensors pick up the light emitted or reflected by eachparticle as the laser hits them.
The sensors transmit the data to a computer where it canbe analyzed.
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What does the data say?
Thats what were trying to find out!
Flow Cytometry data can showmedical researches whether newcures are having an affect, whether
a person has AIDS or not, whether aperson is rejecting a transplantedorgan, and many other things inother fields as well
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How do we analyze the data?
By using software called FlowJo
FlowJo allows you to analyze raw data from a flowcytometer graphically and numerically.
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Setting parameters
Different parameters (the variables on a graph) tell you
different things
For example, forward scatter indicates size of the cell,while side scatter indicates granularity (how much stuff is
inside)
Other parameters that you can observe on a graph includethe different fluorescent markers used to stain cells
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Each fluorescent marker used becomes a parameter in
FlowJo
Graphing two fluorescent markers against each other cantell you which parts of the data were positive for one, both, orneither of the markers
Since certain types of cells bind to certain kinds of markers,this shows you what kind of cells they are
Fluorescent markers as
parameters
Remember this?
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Amount of Blue Markers
A
mountofYe
llow
Marker
s
Remember this?
12
3 4
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BUT
Again, most real-world cytometrysamples contain thousands of cells, not
just four In these cases, you can look at areas of
greater density to identify different types
of cells Lets see that example again:
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Forward Scatter (ForSc)
S
ideScatter
(OrthSc)
Each one of these dotsrepresents a single cell!
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Histograms
There are manydifferent types ofgraphs in Flow
Cytometry softwarelike FlowJo
A histogram is aspecial type of graphthat shows the
frequency of cellsalong the spectrum ofa given parameter
QuickTime and a
decompressorare needed to see this picture.
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Pseudocolor
This is usually thedefault view forsamples
http://www.flowjo.com
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Contour Plots
These are probabilitycontour plots that oftenresemble topographical
maps These will usually be
the best display option
Their biggest downsideis that they do not
usually display outliers,however, in FlowJothere is an option todisplay outliers
http://www.flowjo.com
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Suggested Day 5
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Intro to Gating
Candy sorting activity!
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More gating
Like you saw in the activity you just did, cells can beseparated into different subgroups, while remaining
part of the larger group.
This is very handy, as you can gate a group with oneset of parameters, and then gate the subgroups withdifferent parameters. An example of this is shown onthe next slide.
SampleSample/singlets
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QuickTime and a
decompressorare neede d to see this picture. QuickTime and a
decompressorare needed to see this picture.
QuickTime and a
decompressorare needed to see this picture.
QuickTime and a
decompressorare needed to see this picture.
Sample/singlets/CD3/CD4+ Sample/singlets/CD3
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Statistical analysis
Once weve gated the data, FlowJo has
some handy tools for doing statisticalanalysis.
FlowJo can compute median, mean,geometric mean, mode, and many otherthings.
FlowJo can also find whats calledFrequency of Parent, Grandparent, or Total,which is basically the percent the subgroupis of one of the groups above it.
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Another screenshot here
QuickTime and a
decompressorare needed to see this picture.
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This is analysis in very basic
terms.
In reality, it is much more complicated.
There can be hundreds of parameters and controlsin a single sample, and many samples perexperiment.
It is also possible to find out much more than justthe types of cells in the experiment; however, its
really hard to understand and ever harder to do.
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Good Information Resources
http://www.youtube.com/watch?v=nAfL4FXju1s
http://www.abdserotec.com/uploads/Flow-Cytometry.pdf
http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/
http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/
http://www.unsolvedmysteries.oregonstate.edu/flow_04
http://www.unsolvedmysteries.oregonstate.edu/flow_06
Wikipedia.org
http://www.youtube.com/watch?v=nAfL4FXju1shttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.unsolvedmysteries.oregonstate.edu/flow_04http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_06http://www.unsolvedmysteries.oregonstate.edu/flow_04http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.bdbiosciences.com/immunocytometry_systems/support/training/online/ITF/http://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.abdserotec.com/uploads/Flow-Cytometry.pdfhttp://www.youtube.com/watch?v=nAfL4FXju1s7/31/2019 Physics Presentation(1)
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Image Resources
http://www.Abdcerotec.com
http://www.scq.ubc.ca/flow-cytometry-a-technology-to-count-and-sort-cells/
http://www.bdbiosciences.com
http://www.antonine-education.co.uk/
http://www.plaza.ufl.edu
http://cyto.perdue.edu/Abdcerotec.com
http://www.flowjo.com