Courses in Flow Cytometry

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Courses in Flow Cytometry

• Nucleic Acid Analysis/Cell Cycle Analysis

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Goals of presentation

• Introduction to a few of the most common nucleic
  acid dyes.
• Make researcher aware that there are many
  specific nucleic acid analysis applications that
  are possible with flow cytometry.
• Make researcher aware of common problems
  associated with cell cycle analysis.
• Proper cell cycle protocol

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Advantages to flow cytometric DNA analysis.

• Ethanol fixation allows cells to be harvested and
  fixed at defined time points and to be analyzed
  at a later time.
• Many surface antigens are resistant to ethanol
  fixation, so that DNA analysis can be combined
  with standard immunofluorescent techniques.
• DNA content provides information about ploidy and
  cell cycle distribution.
• Alternatively, cellular RNA content characterizes
  cell phenotypes associated with differentiation,
  quiescence, and proliferation.
• Parrafin-embedded tissues allows for
  retrospective studies.

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General Outline

• Section I
• Nucleic Acid Dyes
• Section II
• Most common nucleic acid analysis applications
• Section III
• Cell cycle analysis with PI

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Section I

• Nucleic Acid Dyes

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How do you know what dye to use?

• Characteristics of dyes
• Spectral properties
• Excitation of the dye. Do you have access to the
  required laser? UV? 488? 633?
• Chemical properties
• Binding characteristics.
• Dyes with base pair specificity cant be used to
  compare genome sizes of different species.
• Also, early in DNA synthesis AT-rich regions are
  replicated first followed by CG-rich regions
  later in S phase. Therefore different DNA dyes
  will give different cell cycle profiles.

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Requirements for a dye to be useful for the
quantitation of DNA and RNA on a per cell basis

• The dye needs to be specific for nucleic acids
  and nothing else
• The dye should exhibit a reasonable degree of DNA
  or RNA selectivity.
• After staining, emission form the dye should be
  stoicheometric with either the cellular DNA or
  RNA content.
• Ideally, a nucleic acid stain should show a
  strong degree of fluorescence enhancement upon
  binding to its nucleic acid target.

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Nucleic acid dyes fall into two basic catagories.

• Base pair binding dyes
• DAPI
• Hoechst 33342
• Hoechst 33258

• Intercalating dyes
• 7-AAD
• PI
• Ethidium bromide
• Acridine Orange
• Pyronin Y

• And many more!!!!

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DNA minor groove-binding

• These dyes bind exclusively to the minor groove
  of double stranded DNA. This gives these dyes
  selectivety for DNA only.
• Hoechst dyes
• 33342- Permeant, for live cells, binds minor
  groove at stretches of at least three AT base
  pairs flanked by one GC base pair
• 33258- Impermeant, binds minor groove at
  stretches of at least three AT base pairs flanked
  by one GC base pair
• DAPI

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Intercalating dyes

• These dyes intercalate between bases of DNA and
  RNA
• PI, no base pair selectivety, impermeant
• Ethinium bromide, no base pair selectivety,
  impermeant
• 7-AAD, slight GC selectivity, impermeant
• Dimeric cyanine dyes
• Intercalating dyes that express different
  emission spectra depending on whether DNA or RNA
  is bound.
• The Acridines- ds nucleic acid gives rise to
  emission at 530nm, ss nucleic acid gives rise to
  emmission at 640nm
• Pyronin Y- No base pair specificity

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The complexity of the binding modes of dyes calls
for careful control of staining conditions.

• To determine to correct staining time- take a
  known amount of cells and a known amount of dye.
  Then analyze on a flow cytometer. When the
  histogram peak no longer moves, that is the
  preferred staining time.

PI labelled Nuclei
Incubated additional 10 min _at_ 37 degrees
Poorly stained
Properly stained
Taken from Purdue University Cytometry
Laboratories and modified by James Marvin
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Summary of Section I

• With any given application, there exists a
  number of dyes that can be used.
• Become familiar with the chemical, spectral, and
  binding properties of the dye being used.

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Section II
What is the right Nucleic Acid detection method
for you

• DNA content
• Subset of cells
• Apoptosis
• Kinetics of proliferation
• Cell cycle analysis

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Determining DNA Content

• DNA binding dye with appropriate reference
  standard
• PI,DAPI,EB with trout or chicken RBCs
• Measure Peak

2C Sample MFI 30
225/3010pgms/Xpgms
CRBC MFI225
4C Sample MFI60
CRBC10pgms
X1.34pgms
8C Sample MFI120
Taken from Current Protocols in Cytometry and
modified by James Marvin
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Determining the ploidy of the cells

• PI, DAPI, EB with appropriate reference standard

Aneuploid tumor cell nuclei
CRBSs
Trout erythrocytes
Diploid normal nuclei
Taken from Current Protocols in Cytometry and
modified by James Marvin
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Ploidy controls
Diploid control alone
Diploid control mixed with tissue sample
Tissue sample alone
Taken from Current Protocols in Cytometry and
modified by James Marvin
Hypoploidy
Hyperploidy
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Subset of cells of interest, proliferating or not?

• Surface marker plus PI or Hoechst

Created by Julie Auger and modified by James
Marvin
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DNA analysis as an indicator of apoptosis.
G0,G1
Apoptotic cells
of cells
S
G2,M
PI (DNA Content)
In addition to DNA analysis, one could also
distinguish apoptotic cells with a variety of
different detection methods. PLEASE inquire if
interested.
Taken from Purdue University Cytometry
Laboratories and modified by James Marvin
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The Cell Cycle
Taken from James Leary and modified by James
Marvin
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What is cell cycle telling us.

• Measurement of cellular DNA content can give an
  estimate of each phase of the cell cycle,
• Also its a measurement of the growth
  characteristics of a cell line or tissue under
  normal or stress conditions.

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Separating different stages of the cell cycle

• Differential staining of DNA and RNA
• Acridine Orange
• Current Protocols in cytometry Section 7.3
• BrdU incorporation
• Section 7.7
• Cyclin analysis
• Section 7.9

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Acridine Orange

• Separates G0 from G1

Taken from Current Protocols in Cytometry and
modified by James Marvin
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Mitotic cells- Histone H3-P
Reacts with cells from prophase to
telophase, weaker in interphase
Juan et al
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Cyclin analysis

• Based on cell cycle
• Dependant on expression of cyclin proteins

Cyclinsare a class of gene products which control
the transition of cells from one cell cycle phase
to another. In normal cells these control points
are predictable. In perturbed or tumor cells
these relationships are changed, frequently
leading to uncontrolled growth Cyclin
Cell cycle phase cdk Protein
Localization A
S and G2/M cdc2/cdk1,cdk2
Nucleus B1 G2/M
cdc2/cdk1
cytoplasm B2 G2/M
cdc2/cdk1
cytoplasm B3 G2/M
cdc2/cdk1,cdk2
Nucleus D1 G1
dk4/cdk6/cdk2
Nucleus D2 G1
ND
Nucleus D3 G1
cdk4/cdk6
Nucleus E G1/S
ND
Nucleus H All phases
CDK7
ND
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Expression of several cyclins throughought the
cell cycle
D(1,2,3)
Taken from Current Protocols in Cytometry and
modified by James Marvin
B1
A
E
Tumor cells show abnormal or inappropriate
expression of these cyclins at these points in
the cell cycle
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Cyclin expression at different stages of the cell
cycle
Taken from Current Protocols in Cytometry and
modified by James Marvin
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Brdu incorporation

• Because of the need for double stranded DNA for
  content labeling and the need for denatured DNA
  for detection of BrdU, specific sample
  preparation guidelines most be empirically
  determined for each cell type

Taken from Current Protocols in Cytometry and
modified by James Marvin
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What are the kinetics of your cell population?

• BrdU incorporation
• Pulse and chase experiment

BrdU expression
Taken from Current Protocols in Cytometry and
modified by James Marvin
DNA Content
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Determining rough estimates of how many cells are
in G0/G1, S, G2/M phase?

• PI, DAPI, EB, for fixed cells
• Divide histogram into three sections
• Hoechst 33342 staining for live cells

G0,G1
G2,M
S
DNA Content
Taken from Purdue University Cytometry
Laboratories and modified by James Marvin
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Summary of Section II

• Be aware that with flow cytometry there are many
  capabilities associated with Nucleic acid
  analysis.
• Make sure that the application you chose is best
  fitted for your experiment.
• Ie. Will you receive the most meaningful data
  possible?

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Section III

• Cell cycle analysis with PI

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Quality Control for Nucleic acid analysis

• Controls
• Narrow cvs
• Should form doublets and triplets
• Should be large as possible
• Should contain true cycling cells
• Staining procedure must be tightly regulated
• Residual dye in tubing can skew data
• Data Analysis

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Effect of CVs on cell cycle
Created by James Leary modified by James Marvin
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Sample preparation

• There are modeling programs that include
  background debris subtraction, however best
  results are received when dead cells are removed
  by centrifuging with F/H
• Make sure that all reagents are DNase free ie.
  Boil for at least 15 minutes

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Cell cycle analysis with PI

• Protocol
• Sample preparation
• Doublet discrimination
• Data analysis

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Cell cycle protocol with PI

• Harvest cells-wash 2X in PBS to get rid of serum
  proteins.
• Resuspend pellet in PBS (up to 36 cells in 1.2
  mls)
• Make sure PBS is Ca and Mg free. Ca and Mg in
  the PBS will cause the cells to agglutinate.
• Add 3.0 ml 95 ethanol dropwise while vortexing.
• Fix in this final 70 ethanol solution for at
  least 30 min. The cells can remain in this
  solution for up to one week.
• Wash cells 2X in PBS in a total volume of 15ml.
  Spin at 2000-2200rpm for 10 min per spin.
  Pelleting cells out of ethanol is more difficult
  and requires a harder spin. If this is not done,
  this step can account for a dramatic loss of
  cells.
• Resuspend pellet in 4.5ml PBS. Add .5 ml RNase
  stock. Incubate for 30 min at 37C.
• Wash 2X in PBS.
• Count cells
• Resuspend in .5-2.0 ml PI stain solution (final
  concentration of 1X106 cells/ml) incubate for
  30 min at 4C or on ice.
• Analyze

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Summary of Doublet Discrimination

• The definition of a doublet (for this
  presentaion) is defined as two G0/1 cells stuck
  together as they traverse the laser.
• The cytometer processes the pulse as one event
  because the pulse that is generated never drops
  below a set threshold level.
• Thus two G0/1 cells will have a similar pulse
  height as a G2/M cell.
• This leads to an incorrect overestimate of cells
  that are G2/M.
• Although a G2M cell has twice the volume of a
  G0/1 cell, diameter only increases by 26.
• On the other hand, the combined diameter of a
  G0/1 doublet is TWICE that of a single G0/1
  event, provided that hydrodynamic focusing aligns
  the cells in the direction of flow
• Therefore, the width to area ratio, which is an
  measurement total fluorescence and length of time
  it takes the the cells to traverse the laser
  beam, increases at a disproportionate value with
  a doublet than with an actual G2 cell.
• Therefore the analysis of pulse width makes it
  possible to find the doublets.

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The Voltage Pulse

• As a cell passes through the laser, more and more
  fluorescent light is emitted until the cell is in
  the center of the laser (maxima)
• As the cell leaves the laser, less and less
  fluorescent light is emitted
• And since emitted photons are converted to
  photoelectrons in the PMT, this creates a voltage
  pulse

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The Pulse
Created by Ryan Duggan
Above threshold
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Measurements of the Pulse
Voltage Intensity
Time
Created by Ryan Duggan
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Measurement of a Doublet pulse
Threshold
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Single Go pulse
Single G2 pulse
Doublet pulse
VS
VS
Width of pulse
Width of pulse
WidthW
WidthW(W.26)
Width2W
What do these pulses show? 1.Width of single Go
and G2 is almost the same 2.Height of G2 and
doublet is about the same 3. If you only look at
pulse height, the G2 cell can not be
differentiated from the doublet.
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Instrument setup
SSC
FL-2A
FL-2W
FSC
No RNase
M1
M2
M3
FL-2A
FL-2A
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Summary of Section III

• The better the sample preparation the more
  meaningful your data will be.
• Most common sources of error associated with cell
  cycle analysis include
• DNases in solutions
• Not adding Ethanol dropwise while vortexing
• Didnt add RNase
• Loss of cells during wash steps, especially when
  spinning out of the ethanol fixing solution
• Doublet discrimination is very important to
  eliminate false G2,M cells.

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Data analysis

• Cell quest
• Modfit
• WinList
• WinCycle
• Flowjo

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Cellquest vs Modfit
M1G0-G1
M3G2-M
M2S
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Works Cited

• Leary, J., http//stem.utmb.edu/98pth6311
• Current Protocols in Cytometry