Courses in Flow Cytometry Nucleic Acid Analysis/Cell Cycle Analysis

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.Make researcher aware of common problems associated with cell cycle analysis.Proper cell cycle protocol

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

Section I Nucleic Acid Dyes

Section II Most common nucleic acid analysis

applications

Section III Cell cycle analysis with PI

Section I

Nucleic Acid Dyes

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

The dye needs to be specific for nucleic acids and nothing elseThe 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

•And many more!!!!

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 dyes33342- Permeant, for live cells, binds minor groove at stretches of at least three AT base pairs flanked by one GC base pair33258- Impermeant, binds minor groove at stretches of at least three AT base pairs flanked by one GC base pairDAPI

Intercalating dyesThese 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.

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

Poorly stained Properly stained

Incubated additional 10 min @ 37 degrees

Taken from Purdue University Cytometry Laboratories and modified by James Marvin

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.

DNA contentSubset of cells ApoptosisKinetics of proliferationCell cycle analysis

Section II

What is the right Nucleic Acid detection method for you

Determining DNA ContentDNA binding dye with appropriate reference standard

PI,DAPI,EB with trout or chicken RBC’sMeasure Peak

CRBC MFI=225CRBC=10pgms

2C Sample MFI= 304C Sample MFI=60

8C Sample MFI=120

225/30=10pgms/XpgmsX=1.34pgms

Taken from Current Protocols in Cytometry and modified by James Marvin

Determining the ploidy of the cells

PI, DAPI, EB with appropriate reference standard

Aneuploid tumor cell nuclei

Trout erythrocytes

Diploid normal nuclei

CRBS’s


Ploidy controlsDiploid control alone

Diploid control mixed with tissue sample

Tissue sample alone

Hyperploidy

Hypoploidy


Subset of cells of interest, proliferating or not?

FL3-W

FL3-A

R1

R2

R3

CD

4

FL3-A

3.89%

Gated on R1 and R3= CD4 negative

12.03%

Gated on R1 and R2= CD4 positives

Surface marker plus PI or Hoechst

Created by Julie Auger and modified by James Marvin

DNA analysis as an indicator of apoptosis.

Apoptotic cells

G0,G1

S G2,M

PI (DNA Content)

# o

f ce

lls

In addition to DNA analysis, one could also distinguish apoptotic cells with a variety of different detection methods. PLEASE inquire if interested.


G1(Gap1) specific regions of the genome become accessible to RNA polymerases. RNA and protein synthesis resume at a rapid rate.

Necrosis due t cell injury can occur at any cell cycle stage

S-phase(DNA synthesis) High rate of synthesis of AT-rich DNA early in S-phases, high rate of synthesis of GC rich DNA late in S-phase

Apoptosis

G2 (Gap 2) Chromosome condensation occurs

G0(noncycling, Quiesnent cells)

The Cell Cycle

M (Mitosis) Nuclear membrane disappeas; homoloques of each chromosome pair pulled to opposite polls of cell; at end of mitosis the cell membrane pinches off to form 2 daughter cells and completes cytokinesis

Taken from James Leary and modified by James Marvin

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

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

DNA Content

RN

A C

on

ten

t

Separates G0 from G1


Mitotic cells- Histone H3-P

Reacts with cells from prophase to telophase,weaker in interphase

Juan et al

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 growthCyclin Cell cycle phase cdk Protein LocalizationA S and G2/M cdc2/cdk1,cdk2 NucleusB1 G2/M cdc2/cdk1 cytoplasmB2 G2/M cdc2/cdk1 cytoplasmB3 G2/M cdc2/cdk1,cdk2 NucleusD1 G1 dk4/cdk6/cdk2 NucleusD2 G1 ND NucleusD3 G1 cdk4/cdk6 Nucleus E G1/S ND NucleusH All phases CDK7 ND

Expression of several cyclins throughought the cell cycle

A

B1

E

D(1,2,3)

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


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


What are the kinetics of your cell population?

DNA Content

Brd

U e

xp

ress

ion

BrdU incorporation Pulse and chase experiment


S

G0,G1 G2,M

DNA Content

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


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

Cell cycle analysis with PI

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 regulatedResidual dye in tubing can skew dataData Analysis

Effect of CV’s on cell cycle

CV=2You wish

CV=5Upper end of CV’s for good cell cycle analysis

CV=8Only for live cells and you are desparate

CV=15Don’t even try

Created by James Leary modified by James Marvin

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/HMake sure that all reagents are DNase free ie. Boil for at least 15 minutes

Cell cycle analysis with PI

Protocol Sample preparationDoublet discriminationData analysis

Cell cycle protocol with PIHarvest 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 cellsResuspend 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 DiscriminationThe 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 flowTherefore, 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

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

TimeTime

FL-2

Heig

ht

FL-2

Heig

ht

det e

ctor

dete

ctor

Above threshold

Created by Ryan Duggan

Measurements of the Pulse

Pulse Height

Pulse Width= time of flight

Pulse Area

TimeTime

Vol

tage

Int

ensi

tyV

olta

ge I

nten

sity

Created by Ryan Duggan

TimeTime

FL-2

Heig

ht

FL-2

Heig

ht

det e

ctor

dete

ctor

Threshold

Measurement of a Doublet pulse

Width of pulseVSWidth of pulse

Single Go pulse Doublet

pulse

Width of pulse

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.

Single G2 pulse

VS

Width=W Width=W+(W*.26)

Width~2W

Instrument setup

FL-2AFL-2A

FL-2

A

FL-2WFSC

SSC

No RNaseM1

M2

M3

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 analysisCell questModfitWinListWinCycleFlowjo

Cellquest vs Modfit

M2=SM3=G2-M

M1=G0-G1

Works Cited

Leary, J., http://stem.utmb.edu/98pth6311Current Protocols in Cytometry

Documents

Courses in Flow Cytometry Nucleic Acid Analysis/Cell Cycle Analysis