Courses in Flow Cytometry

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

2. 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

3. 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.

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

5. Section I • Nucleic Acid Dyes

6. 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.

7. 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.

8. 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!!!!

9. 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

10. 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

11. 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

12. 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.

13. Section II What is the right Nucleic Acid detection method for you • DNA content • Subset of cells • Apoptosis • Kinetics of proliferation • Cell cycle analysis

14. 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

15. 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

16. 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

17. 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

18. 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

19. The Cell Cycle Taken from James Leary and modified by James Marvin

20. 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.

21. 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

22. RNA Content DNA Content Acridine Orange • Separates G0 from G1 Taken from Current Protocols in Cytometry and modified by James Marvin

23. Mitotic cells- Histone H3-P Reacts with cells from prophase to telophase, weaker in interphase Juan et al

24. 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

25. 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

26. Cyclin expression at different stages of the cell cycle Taken from Current Protocols in Cytometry and modified by James Marvin

27. 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

28. 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

29. 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

30. 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?

31. Section III • Cell cycle analysis with PI

32. 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

33. Effect of CV’s on cell cycle Created by James Leary modified by James Marvin

34. 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

35. Cell cycle analysis with PI • Protocol • Sample preparation • Doublet discrimination • Data analysis

36. 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

37. 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.

38. 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

39. Time FL-2 Height detector The Pulse Created by Ryan Duggan Above threshold

40. Pulse Height Pulse Area Pulse Width= time of flight Measurements of the Pulse Voltage Intensity Time Created by Ryan Duggan

41. Time FL-2 Height detector Measurement of a Doublet pulse Threshold

42. 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.

43. Instrument setup SSC FL-2A FSC FL-2W No RNase M1 M2 M3 FL-2A FL-2A

44. 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.

45. Data analysis • Cell quest • Modfit • WinList • WinCycle • Flowjo

46. Cellquest vs Modfit M1=G0-G1 M3=G2-M M2=S

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