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.
DNA content- compare to controls
RNA content- Quiescence, low levels of RNA in Go and increasing levels in G1 through M phase

4. General Outline Section I Section II Section III Nucleic Acid Dyes
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. 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!!!!

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
Permeant, for live cells, binds minor groove at stretches of at least three AT base pairs flanked by one GC base pair
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 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
CRBC=10pgms
4C Sample MFI=60
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. Subset of cells of interest, proliferating or not?
Surface marker plus PI or Hoechst
FL3-W
FL3-A R1
12.03%
Gated on R1 and R2= CD4 positives R2 R3
CD4
FL3-A
3.89%
Gated on R1 and R3= CD4 negative
Created by Julie Auger and modified by James Marvin

18. DNA analysis as an indicator of apoptosis.
G0,G1
# of cells
Apoptotic 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 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
G1(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 occurs
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
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. Acridine Orange Separates G0 from G1 RNA Content DNA Content
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,cdk Nucleus
B G2/M cdc2/cdk cytoplasm
B G2/M cdc2/cdk cytoplasm
B G2/M cdc2/cdk1,cdk Nucleus
D G dk4/cdk6/cdk Nucleus
D G ND Nucleus
D G cdk4/cdk Nucleus
E G1/S ND Nucleus
H All phases CDK 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
BrdU calls for double stranded DNA for labeling with PI and at the same time calls for denatured DNA for labeling with anti-BrdU antibody
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 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
Controls- this will allow for the correct location by channel number of the normal diploid sample

33. Effect of CV’s on cell cycle
Upper end of CV’s for good cell cycle analysis
CV=2
You wish
CV=8
Only for live cells and you are desparate
CV=15
Don’t even try
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 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 cells
Resuspend in 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. The Pulse FL-2 Height detector Time Created by Ryan Duggan
Above threshold
Lets look at a few snapshots of time as the cell is progressing through the laser beam
First, 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.

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

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

42. What do these pulses show?
Single Go pulse
Single G2 pulse
Doublet pulse
Width of 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