Presentation on theme: "Courses in Flow Cytometry"— Presentation transcript:
1Courses in Flow Cytometry Nucleic Acid Analysis/Cell Cycle Analysis
2Goals of presentationIntroduction 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
3Advantages 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.DNA content- compare to controlsRNA content- Quiescence, low levels of RNA in Go and increasing levels in G1 through M phase
4General Outline Section I Section II Section III Nucleic Acid Dyes Most common nucleic acid analysis applicationsSection IIICell cycle analysis with PI
6How do you know what dye to use? Characteristics of dyesSpectral propertiesExcitation of the dye. Do you have access to the required laser? UV? 488? 633?Chemical propertiesBinding 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.
7Requirements 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.
8Nucleic acid dyes fall into two basic catagories. Base pair binding dyesDAPIHoechst 33342Hoechst 33258Intercalating dyes7-AADPIEthidium bromideAcridine OrangePyronin YAnd many more!!!!
9DNA minor groove-binding These dyes bind exclusively to the minor groove of double stranded DNA. This gives these dyes selectivety for DNA only.Hoechst dyesPermeant, for live cells, binds minor groove at stretches of at least three AT base pairs flanked by one GC base pairImpermeant, binds minor groove at stretches of at least three AT base pairs flanked by one GC base pairDAPI
10Intercalating dyes These dyes intercalate between bases of DNA and RNA PI, no base pair selectivety, impermeantEthinium bromide, no base pair selectivety, impermeant7-AAD, slight GC selectivity, impermeantDimeric cyanine dyesIntercalating 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 640nmPyronin Y- No base pair specificity
11The 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 NucleiIncubated additional degreesPoorly stainedProperly stainedTaken from Purdue University Cytometry Laboratories and modified by James Marvin
12Summary of Section IWith 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.
13Section II What is the right Nucleic Acid detection method for you DNA contentSubset of cellsApoptosisKinetics of proliferationCell cycle analysis
14Determining DNA Content DNA binding dye with appropriate reference standardPI,DAPI,EB with trout or chicken RBC’sMeasure Peak2C Sample MFI= 30225/30=10pgms/XpgmsCRBC MFI=225CRBC=10pgms4C Sample MFI=60X=1.34pgms8C Sample MFI=120Taken from Current Protocols in Cytometry and modified by James Marvin
15Determining the ploidy of the cells PI, DAPI, EB with appropriate reference standardAneuploid tumor cell nucleiCRBS’sTrout erythrocytesDiploid normal nucleiTaken from Current Protocols in Cytometry and modified by James Marvin
16Ploidy controls Diploid control alone Diploid control mixed with tissue sampleTissue sample aloneTaken from Current Protocols in Cytometry and modified by James MarvinHypoploidyHyperploidy
17Subset of cells of interest, proliferating or not? Surface marker plus PI or HoechstFL3-WFL3-AR112.03%Gated on R1 and R2= CD4 positivesR2R3CD4FL3-A3.89%Gated on R1 and R3= CD4 negativeCreated by Julie Auger and modified by James Marvin
18DNA analysis as an indicator of apoptosis. G0,G1# of cellsApoptotic cellsSG2,MPI (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
19The 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 cytokinesisG1(Gap1) specific regions of the genome become accessible to RNA polymerases. RNA and protein synthesis resume at a rapid rate.G0(noncycling, Quiesnent cells)G2 (Gap 2) Chromosome condensation occursNecrosis due t cell injury can occur at any cell cycle stageS-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-phaseApoptosisTaken from James Leary and modified by James Marvin
20What 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.
21Separating different stages of the cell cycle Differential staining of DNA and RNAAcridine OrangeCurrent Protocols in cytometry Section 7.3BrdU incorporationSection 7.7Cyclin analysisSection 7.9
22Acridine Orange Separates G0 from G1 RNA Content DNA Content Taken from Current Protocols in Cytometry and modified by James Marvin
23Mitotic cells- Histone H3-P Reacts with cells from prophase to telophase,weaker in interphaseJuan et al
24Cyclin analysis Based on cell cycle Dependant on expression of cyclin proteinsCyclinsare 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,cdk NucleusB G2/M cdc2/cdk cytoplasmB G2/M cdc2/cdk cytoplasmB G2/M cdc2/cdk1,cdk NucleusD G dk4/cdk6/cdk NucleusD G ND NucleusD G cdk4/cdk NucleusE G1/S ND NucleusH All phases CDK ND
25Expression of several cyclins throughought the cell cycle D(1,2,3)Taken from Current Protocols in Cytometry and modified by James MarvinB1AETumor cells show abnormal or inappropriate expression of these cyclins at these points in the cell cycle
26Cyclin expression at different stages of the cell cycle Taken from Current Protocols in Cytometry and modified by James Marvin
27Brdu incorporationBrdU calls for double stranded DNA for labeling with PI and at the same time calls for denatured DNA for labeling with anti-BrdU antibodyBecause 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 typeTaken from Current Protocols in Cytometry and modified by James Marvin
28What are the kinetics of your cell population? BrdU incorporationPulse and chase experimentBrdU expressionTaken from Current Protocols in Cytometry and modified by James MarvinDNA Content
29Determining rough estimates of how many cells are in G0/G1, S, G2/M phase? PI, DAPI, EB, for fixed cellsDivide histogram into three sectionsHoechst staining for live cellsG0,G1G2,MSDNA ContentTaken from Purdue University Cytometry Laboratories and modified by James Marvin
30Summary of Section IIBe 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?
32Quality Control for Nucleic acid analysis ControlsNarrow cv’sShould form doublets and tripletsShould be large as possibleShould contain true cycling cellsStaining procedure must be tightly regulatedResidual dye in tubing can skew dataData AnalysisControls- this will allow for the correct location by channel number of the normal diploid sample
33Effect of CV’s on cell cycle Upper end of CV’s for good cell cycle analysisCV=2You wishCV=8Only for live cells and you are desparateCV=15Don’t even tryCreated by James Leary modified by James Marvin
34Sample preparationThere 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
35Cell cycle analysis with PI ProtocolSample preparationDoublet discriminationData analysis
36Cell 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 rpm 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 ml PI stain solution (final concentration of 1X106 cells/ml) & incubate for 30 min at 4C or on ice.Analyze
37Summary 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 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.
38The Voltage PulseAs 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 emittedAnd since emitted photons are converted to photoelectrons in the PMT, this creates a voltage pulse
39The Pulse FL-2 Height detector Time Created by Ryan Duggan Above thresholdLets look at a few snapshots of time as the cell is progressing through the laser beamFirst, when the cell has yet to reach the laser, no light is scattered and the window of time remains closed.As the cell enters the laser beam. It scatters some light. If the threshold is surpassed, the window of time is opened and the voltage is recorded as time passes.As the cell reaches the center of the beam, a maximum amount of light is scattered, and the maximum voltage is recorded.As the cell leaves the the laser beam, the voltage decreases. After the set amount of time has expired, the window closes until the threshold is surpassed again.
40Measurements of the Pulse Pulse HeightPulse AreaPulse Width=time of flightVoltage IntensitySince the voltage pulse is proportional to the amount of photons reaching the detector, by measuring the pulse, we are given information as to how much light was emitted or scattered from the cell. There are 3 main measurements of the pulse.Pulse height will give you the maximum amount of fluorescence.Pulse width will give you information as to how long the cell was in the laser beam. This information is useful when discriminating against doublets (or two cells stuck together). If two cells come in to the laser beam together, than they will have a longer width. So by looking at the width of a signal, one can chose to only look at signal cells and gate out doublet or aggregates.Finally you can measure the Pulse Area. This value will give you the total fluorescence of the cell as opposed to just the maximum value.TimeCreated by Ryan Duggan
41Measurement of a Doublet pulse TimeFL-2 Height detectorThreshold
42What do these pulses show? Single Go pulseSingle G2 pulseDoublet pulseWidth of pulseVSVSWidth of pulseWidth of pulseWidth=WWidth=W+(W*.26)Width~2WWhat do these pulses show?1.Width of single Go and G2 is almost the same2.Height of G2 and doublet is about the same3. If you only look at pulse height, the G2 cell can not be differentiated from the doublet.
44Summary of Section IIIThe better the sample preparation the more meaningful your data will be.Most common sources of error associated with cell cycle analysis include;DNases in solutionsNot adding Ethanol dropwise while vortexingDidn’t add RNaseLoss of cells during wash steps, especially when spinning out of the ethanol fixing solutionDoublet discrimination is very important to eliminate false G2,M cells.