An Introduction to Flow Cytometry By Max Parker-Shames

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An Introduction to Flow Cytometry

• By
• 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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But First, Some ReviewImmunology 101
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The Immune System

• A layered cake

• Responds to pathogens (bacteria, viruses, or
  other microorganisms that cause disease) and
  works to keep your body healthy
• It has to distinguish between the bodys cells
  and foreign cells
• There are multiple layers of defense in the body,
  like the layers of a cake

becomehealthynow.com/... immune_home.jpg
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Surface Barriers
lestout.com/ ... article-skin-nonspecific-immune-s
ystem-defence.jpg

• Physical, i.e. skin, coughing/sneezing, etc
• These are innate they are non-specific and
  general defenses
• generic response
• no long-lasting immunity
• includes inflammation (a reaction of the
  vascular system whereby chemicals from white
  blood cells are released from the blood stream)
• the complement system (enzymes and proteins found
  in the blood)
• and cellular barriers (cell membranes)

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White Blood Cells

• Cells that defend against disease and foreign
  materials
• A high number of white blood cells can indicate
  disease
• Some Different Kinds
• Neutrophils defend against bacterial and fungal
  infections
• Eosinophils deal with parasitic infections
• Basophils responsible for allergic/antigen
  response (release histamine)
• Lymphocytes 3 main types
• B-cells
• T-cells
• Natural killer cells
• Monocytes work with T-cells to help recognize
  pathogens

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Lymphocytes
www. thehumanbody.ecsd.net
A type of White Blood Cell responsible for immune
response

• B-cells
• make antibodies
• T-cells
• responsible for immunological memory
• Includes CD4 cells and CD8 cells
• Natural killer cells
• carpet-bomb the area where it thinks the disease
  is
• The cause of sore throat/achiness when sick

Antibody - a protein designed to identify a
certain type of cell
Antigen - what an antibody attaches to (the
pathogen)
Epitope - the thing on the antigen that is
recognized by the immune system and allows the
antibody to attach
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Proteins
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Structure

• Amino acids linked with peptide bonds to form
  polypeptides
• The peptide consists of a regularly repeating
  backbone and variable side chains

aloeveraibs.com/wp-content/uploads/2008/08/aminoac
idstruct.jpg
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Levels of Structure

• There are different layers of protein structure
  as each level interacts with itself to create new
  shapes
• There are also two general categories
• Fibrous
• Globular

matcmadison.edu/biotech/resources/protiens/labManu
al/images/220_04_114.png
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Uses of Proteins
cellbiology.med.unsw.edu.au/units/images/Cell_memb
rane.png

• Many proteins are enzymes, which catalyze
  chemical reactions
• They help with metabolism, cell structure,
  cycles, signaling, and immune response
• Most are membrane proteins as part of the
  phospholipid bilayer. Cell recognition proteins
  allow cells to recognize each other and interact

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HIV/AIDS

• An application of Flow Cytometry

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What is it?

• Acquired Immune Deficiency Syndrome or Acquired
  Immunodeficiency Syndrome (AIDS)
• A disease caused by the Human Immunodeficiency
  Virus (HIV) that weakens the immune system
• The disease can actually insert its own genetic
  material into our Chromosomes

teenaids.org/Portals/0/Images/whatisAIDS-pic3.gif
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How does it affect the immune system?

• HIV infects important immune system cells like
  CD4 cells (a type of T-Cell)
• It does so by recognizing a surface protein on
  the CD4 cell
• When it has killed a certain level of CD4 cells,
  it is considered AIDS
• This can take 9 to 10 years

humanillnesses.com/original/images/hdc_0001_0001_0
_img0009.jpg
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Flow Cytometry

• A machine that looks at the cells we just learned
  about

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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 are machines that measure
  multiple aspects of single cells
• The cells are interrogated (examined) by lasers
• They are interrogated as they flow by in a stream
  of fluid.

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What are Flow Cytometers used for?

• Medical Research (especially in cancer research
  and immune functions)
• Medical Diagnoses (For detecting or monitoring
  diseases like HIV/AIDS or for monitoring
  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 translated into
  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.oregonstate.edu/flow_
  06

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Immunophenotyping

• The basis of flow cytometry
• Immunophenotyping is identifying molecules by
  using special antibodies that bind specifically
  to those molecules.
• These antibodies are fluorescent (they emit a
  photon when stimulated)
• In flow cytometry, cells are stained with a
  number of fluorescent antibody dyes, and when the
  laser hits them, they fluoresce at different
  wavelengths.

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Requirements of a good antibody
pleiad.umdnj.edu
Specificity the antibody only binds to antigen
you want it to Affinity irreversible binding
(so the marker doesn't come off) Sensitivity
must tend to bind with the antigen
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Conjugation

• How a fluorochrome attaches to an antibody
• Conjugation allows flow cytometers to detect the
  different cells
• Most reagents are already conjugated when
  purchased

fluorochrome (aka fluorophore) a piece of
molecule that causes molecule to be fluorescent
(emit photon with stimulus)
This is a picture of a flourophore-labeled human
cell
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Requirements of a good fluorescent molecule

• High brightness
• Small amount of excitation gives off a large
  of photons
• Can be detected by sensors in flow cytometer
• Absorption spectrum can be efficiently excited
• Emission spectrum can be efficiently measured
• Certain combinations of fluorochromes for
  multi-color experiments certain combinations
  work better with certain antibodies

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Requirements Cont.

• Consider amount of antigen
• if a lot, use dimmer fluorochrome
• if a little, use brighter fluorochrome
• Doesn't effect the viability of the cell (esp.
  if that's what you're measuring)
• Doesn't interfere with other fluorochromes/dyes
• Isn't destroyed by light exposure while you're
  doing the experiment (photobleaching)

umsl.edu
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Tandem Dyes

• Two fluorescent molecules attaches to each other-
  one gets excited and transfers that energy to the
  other, which then fluoresces at a different
  wavelength.
• Not naturally occuring.
• Useful because they allow detection in different
  parts of the spectrum, thus allowing more options
  from each laser.
• Problems
• Can get a photon 'leak' from the donor.
• Hard to store (photosensitive).
• Tandem lots have different spectral properties,
  so compensation is different for different
  antibodies, adding another layer of complexity.

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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 when
  stimulated 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)
Wikipedia.org
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Amount of Yellow Markers
Amount of Blue Markers
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BUT

• In real-world examples, the graphs will look a
  lot more complicated.
• Most cytometry samples contain thousands 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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Side Scatter (OrthSc)
Each one of these dots represents a single cell!
Forward Scatter (ForSc)
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Cell Sorting

• The Fluidic System

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Fluidic System

• In order to do analyses in Flow Cytometers, the
  machine needs to analyze cells individually
• To accomplish this, cells are run through the
  machine in fast-moving stream of fluid (hence the
  name flow cytometry)
• This process is called Hydrodynamic Focusing

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• The sample fluid stream is directed through the
  laser by the sheath 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 interrogation point

High sample pressure, high average cell count.
This gives less accuracy, but is much faster
Low sample pressure, low average cell count. This
gives greater accuracy, but takes longer
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The Optics System

• Excitation

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Excitation

• Multiple Lasers are used in Flow Cytometers to
  excite the cells
• Fluorescent markers absorb and reemit different
  wavelengths
• Different types of cells scatter different
  colored light, this helps identify what kind of
  cells they are

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Light Sorting

• 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

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Electrostatic Flow Sorting

• After the cells are interrogated by the laser,
  vibrations separate the sample stream into
  droplets containing either one or zero cells,
  called the break-off point

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• At the point at which the stream breaks into
  droplets, it passes through an electrically
  charged ring which charge cells based on the
  results detected by the cytometers laser and
  detector system
• The cells then pass by charged plates which sort
  the cells based on the charge that they have been
  given

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Calibration Quantum (Q) Dots

• Highly fluorescent, nanometer sized crystals that
  you can use to label, like other fluorochromes
• They have very limited spectral overlap (meaning
  that each color looks distinct and does not emit
  overlapping wavelengths)

www.ceac.aston.ac.uk

• Usually used as controls to calibrate flow
  cytometers for fluorescent emission
• Expensive

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Titration

• Titration Optimizing a desired outcome
• How much reagent do you need?
• Flow cytometry users must figure out how much
  reagent per millions of cells to use
• The problem is that manufacturers often say you
  need to use more reagent than you really do, so
  you have to run experiments to find the right
  proportion
• Saves
• Improves accuracy
• Avoids non-specific binding caused by high
  concentration of reagent.

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Lets review what weve learned and look at how
the process works

• T-Shirt sorting activity!

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Electronics System

• So what happens after the cells are interrogated
  by the laser and sorted by type?

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What does the electronics system do?

• Converts the reflected light into electrons
• Converts analog signals 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 to computer
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Compensation

• A very important function of the electronics
  system is to perform compensation

• There is some overlap between the colors emitted
  by different fluorescent markers, therefore
  mathematical compensation is used to reduce
  overlapping results

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Heres a video overview of Flow Cytometry

• http//www.youtube.com/watch?vnAfL4FXju1s

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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
  each particle as the laser hits them.
• The sensors transmit the data to a computer where
  it can be analyzed.

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What does the data say?
Thats what were trying to find out!
Flow Cytometry data can show medical researches
whether new cures are having an affect, whether a
person has AIDS or not, whether a person is
rejecting a transplanted organ, and many other
things in other 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 flow
  cytometer 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
  include the different fluorescent markers used to
  stain cells

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Fluorescent markers as parameters

• Each fluorescent marker used becomes a parameter
  in FlowJo
• Graphing two fluorescent markers against each
  other can tell you which parts of the data were
  positive for one, both, or neither of the markers
• Since certain types of cells bind to certain
  kinds of markers, this shows you what kind of
  cells they are

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Remember this?
1
Amount of Yellow Markers
2
3
4
Amount of Blue Markers
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BUT

• Again, most real-world cytometry samples contain
  thousands of cells, not just four
• Some cells fall in between areas of positive and
  negative traits. This is sometimes in part
  because they undergoing the process of
  differentiation
• Differentiation is the process by which a less
  specified cell (like a stem cell) becomes
  specialized as a specific type of cell

59

• With most flow cytometry graphs with thousands of
  cells, you can look at areas of greater density
  to identify different types of cells
• Lets see that example again

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Side Scatter (OrthSc)
Forward Scatter (ForSc)
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Histograms

• There are many different types of graphs in Flow
  Cytometry software like FlowJo
• A histogram is a special type of graph that shows
  the frequency of cells along the spectrum of a
  given parameter

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Pseudocolor

• This is usually the default view for samples

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Contour Plots

• These are probability contour plots that often
  resemble topographical maps
• These will usually be the best display option
• Their biggest downside is that they do not
  usually display outliers, however, in FlowJo
  there is an option to display outliers

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What are we going to look at in Flow Cytometers?
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Samples

• Often taken from blood, the samples we will be
  looking at focus on white blood cells and T-Cells
• Some of the first basic groups of White Blood
  Cells are monocytes and lymphoctyes

mindoversports.com
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Monocytes and Lymphocytes

• Monocytes circulate in the blood stream and also
  stay in tissues
• They perform phagocytosis, when the cell
  membrane engulfs a solid foreign body
• Lymphocytes include Natural Killer Cells, T Cells
  and B Cells

Wikipedia.org
67
dev.nsta.org/evwebs/1887/immune20system.JPG
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CD4 and CD8

• CD4 is a glycoprotein (a protein containing
  glycan chains) on the surface of Helper T-Cells
  that amplifies the signals sent out by certain
  enzymes. It is also a specific receptor for the
  HIV virus
• CD8 is a glycoprotein that helps bind signaling
  molecules to pathogens

Wikipedia.org
69
Intro to Gating
Candy Sorting Activity!
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More gating

• Like you saw in the activity you just did, cells
  can be separated into different subgroups, while
  remaining part of the larger group.
• This is very handy, as you can gate a group with
  one set of parameters, and then gate the
  subgroups with different parameters. An example
  of this is shown on the next slide.

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Sample
Sample/singlets
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 statistical analysis.
• FlowJo can compute median, mean, geometric mean,
  mode, and many other things.
• FlowJo can also find whats called Frequency of
  Parent, Grandparent, or Total, which is basically
  the percent the subgroup is of one of the groups
  above it.

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(No Transcript)
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So what can all this tell us?

• Using known facts about certain types of cells,
  we can figure out how many and what kind of cells
  there were in the sample.

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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 controls
  in a single sample, and many samples per
  experiment.
• It is also possible to find out much more than
  just the types of cells in the experiment
  however, its really hard to understand and ever
  harder to do.

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Now its time to try your hand at FlowJo!
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Good Information Resources

• http//www.youtube.com/watch?vnAfL4FXju1s
• http//www.abdserotec.com/uploads/Flow-Cytometry.p
  df
• http//www.bdbiosciences.com/immunocytometry_syste
  ms/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

78
Image Resources

• www.wikipedia.com
• mindoversports.com
• www.ceac.aston.ac.uk
• www.thehumanbody.ecsd.net
• dev.nsta.org/evwebs/1887/immune20system.JPG
• lestout.com/ ... article-skin-nonspecific-immune-s
  ystem-defence.jpg
• becomehealthynow.com/... immune_home.jpg
• matcmadison.edu/biotech/resources/protiens/labManu
  al/images/220_04_114.png
• aloeveraibs.com/wp-content/uploads/2008/08/aminoac
  idstruct.jpg
• cellbiology.med.unsw.edu.au/units/images/Cell_memb
  rane.png
• teenaids.org/Portals/0/Images/whatisAIDS-pic3.gif
• humanillnesses.com/original/images/hdc_0001_0001_0
  _img0009.jpg
• pleiad.umdnj.edu/hemepath/immuno/graphics/surf_an.
  gif
• umsl.edu/tsytsarev/... lect10_4.jpg
• Abdcerotec.com
• http//www.scq.ubc.ca/flow-cytometry-a-technology-
  to-count-and-sort-cells/
• ib-bio.wikispaces.com/file/view/cell_differentiati
  on.gif
• http//www.bdbiosciences.com/immunocytometry_syste
  ms/support/training/online/ITF/