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Courses in Flow Cytometry. Nucleic Acid Analysis/Cell Cycle Analysis. 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.

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courses in flow cytometry

Courses in Flow Cytometry

Nucleic Acid Analysis/Cell Cycle Analysis

goals of presentation
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
advantages to flow cytometric dna analysis
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.
general outline
General Outline
  • Section I
    • Nucleic Acid Dyes
  • Section II
    • Most common nucleic acid analysis applications
  • Section III
    • Cell cycle analysis with PI
section i
Section I
  • Nucleic Acid Dyes
how do you know what dye to use
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 can’t 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.
requirements for a dye to be useful for the quantitation of dna and rna on a per cell basis
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.
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

Nucleic acid dyes fall into two basic catagories.
  • And many more!!!!
dna minor groove binding
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
intercalating dyes
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
the complexity of the binding modes of dyes calls for careful control of staining conditions
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 @ 37 degrees

Poorly stained

Properly stained

Taken from Purdue University Cytometry Laboratories and modified by James Marvin

summary of section i
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.
section ii
Section II

What is the right Nucleic Acid detection method for you

  • DNA content
  • Subset of cells
  • Apoptosis
  • Kinetics of proliferation
  • Cell cycle analysis
determining dna content
Determining DNA Content
  • DNA binding dye with appropriate reference standard
    • PI,DAPI,EB with trout or chicken RBC’s
  • Measure Peak

2C Sample MFI= 30

225/30=10pgms/Xpgms

CRBC MFI=225

4C Sample MFI=60

CRBC=10pgms

X=1.34pgms

8C Sample MFI=120

Taken from Current Protocols in Cytometry and modified by James Marvin

determining the ploidy of the cells
Determining the ploidy of the cells
  • PI, DAPI, EB with appropriate reference standard

Aneuploid tumor cell nuclei

CRBS’s

Trout erythrocytes

Diploid normal nuclei

Taken from Current Protocols in Cytometry and modified by James Marvin

ploidy controls
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

subset of cells of interest proliferating or not

Gated on R1 and R2= CD4 positives

R1

FL3-A

12.03%

FL3-W

Gated on R1 and R3= CD4 negative

R2

CD4

3.89%

R3

FL3-A

Subset of cells of interest, proliferating or not?
  • Surface marker plus PI or Hoechst

Created by Julie Auger and modified by James Marvin

dna analysis as an indicator of apoptosis
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

slide19

The Cell Cycle

Taken from James Leary and modified by James Marvin

what is cell cycle telling us
What is cell cycle telling us.
  • Measurement of cellular DNA content can give an estimate of each phase of the cell cycle,
  • Also it’s a measurement of the growth characteristics of a cell line or tissue under normal or stress conditions.
separating different stages of the cell cycle
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
acridine orange

RNA Content

DNA Content

Acridine Orange
  • Separates G0 from G1

Taken from Current Protocols in Cytometry and modified by James Marvin

mitotic cells histone h3 p
Mitotic cells- Histone H3-P

Reacts with cells from prophase to telophase,

weaker in interphase

Juan et al

cyclin analysis
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

expression of several cyclins throughought the cell cycle
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

cyclin expression at different stages of the cell cycle
Cyclin expression at different stages of the cell cycle

Taken from Current Protocols in Cytometry and modified by James Marvin

brdu incorporation
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

what are the kinetics of your cell population
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

determining rough estimates of how many cells are in g0 g1 s g2 m phase
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

summary of section ii
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?
section iii
Section III
  • Cell cycle analysis with PI
quality control for nucleic acid analysis
Quality Control for Nucleic acid analysis
  • Controls
    • Narrow cv’s
    • 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
effect of cv s on cell cycle
Effect of CV’s on cell cycle

Created by James Leary modified by James Marvin

sample preparation
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
cell cycle analysis with pi
Cell cycle analysis with PI
  • Protocol
  • Sample preparation
  • Doublet discrimination
  • Data analysis
cell cycle protocol with pi
Cell cycle protocol with PI
  • Harvest cells-wash 2X in PBS to get rid of serum proteins.
  • Resuspend pellet in PBS (up to 3^6 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
summary of doublet discrimination

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 G2+M 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.

the voltage pulse
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
the pulse

Time

FL-2 Height detector

The Pulse

Created by Ryan Duggan

Above threshold

measurements of the pulse

Pulse Height

Pulse Area

Pulse Width=

time of flight

Measurements of the Pulse

Voltage Intensity

Time

Created by Ryan Duggan

slide41

Time

FL-2 Height detector

Measurement of a Doublet pulse

Threshold

slide42

Width of pulse

Single Go pulse

Single G2 pulse

Doublet pulse

VS

VS

Width of pulse

Width of pulse

Width=W

Width=W+(W*.26)

Width~2W

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.

instrument setup
Instrument setup

SSC

FL-2A

FSC

FL-2W

No RNase

M1

M2

M3

FL-2A

FL-2A

summary of section iii
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
    • Didn’t 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.
data analysis
Data analysis
  • Cell quest
  • Modfit
  • WinList
  • WinCycle
  • Flowjo
cellquest vs modfit

Cellquest vs Modfit

M1=G0-G1

M3=G2-M

M2=S

works cited
Works Cited
  • Leary, J., http://stem.utmb.edu/98pth6311
  • Current Protocols in Cytometry
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