multicycle for windows tutorial

1Phoenix Flow Systems, Inc.

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Provided by

Phoenix Flow Systems, Inc.6790 Top Gun St. #1

San Diego, CA 92121 USA858 453-5095 fax 858 453-2117

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Tutorial Introduction 2

Example 1 - AutoFit analysis of ASCII.2Automatic Fitting of a Two Cell Cycle Histogram 3

Example 2 - ASCII.2Manual Fitting of a Two Cell Cycle Histogram 6

Example 3 - ASCII.3Manual Fitting of Two Cell Cycle Populations from Paraffin 10

Example 4 - ASCII.4Manual Fitting of Three Cell Cycle Populations (One Diploidand Two Aneuploid Peaks) 14

Example 5 - ASCII.1One Cell Cycle and One DNA Peak 17

Example 6 - ASCII.5Synchronized Cultured Cells in S Phase 20

MultiCycle for Windows

TUTORIAL


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TUTORIAL INTRODUCTION

In this tutorial you will learn how to use MultiCycle to analyze different cell cyclehistogram types and file formats. There are six different tutorial examples usingfiles included with MultiCycle for Windows (located in the DEMODATA directory) thatwill illustrate the use of MultiCycle.

If you have no knowledge of performing a DNA cell cycle analysis or need arefresher, we strongly recommend you read the other PDF file installed with theMultiCycle for Windows, “Introduction to Cell Cycle Analysis.”

DNA cell cycle analysis does not have to be difficult. Interpreting the data is thehard part. Cell cycle analysis with MultiCycle for Windows is performed with thesesteps:

1. Select a data file2. Determine the number of total cell cycles in the histogram3. Subtract the debris from the histogram4. Perform the analysis5. Evaluate the results

MultiCycle for Windows has a menu bar right below the main menu, which leadsyou through the above process of performing a cell cycle analysis. Only theappropriate buttons are highlighted at each step of the analysis.

1. Select a data file

2. Determine the number of total cell cycles in the histogram

3. Subtract the debris from the histogramThis will be covered in the tutorial that follows.

4. Perform the analysis and evaluate the results.

Now please take the time to go through each of the tutorial procedures, step by step,so that you will become familiar with MultiCycle for Windows.


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EXAMPLE 1

AUTOMATIC FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOIDAND ANEUPLOID) - ASCII.2

Follow these directions to see how easy it is to perform a complete cell cycle analysis.

STEP 1 Select File ASCII.2

1. Select the menu option [File - Open] from the main menu bar. The contents of thedirectory DEMODATA should be displayed; if not, change the directory in the usualWindows manner looking for \Program Files\Phoenix Flow Systems\MultiCycle forWindows\DEMODATA. If the ASCII file format type has not previously been specified,select [ASCII] on the drop-down Files of Type sub-menu at the bottom of the file openmenu box.

2. From the list of files within this directory, highlight the file [ASCII.2] by clicking overthis name with the mouse button. The file "ASCII.2" is now colored blue, indicating that ithas been chosen.

3. Click the mouse button on [Open] to begin analysis of this file.


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STEP 2 Beginning Automated Cell Cycle Analysis

1. You will see a graph of the histogram contained in the file ASCII.2. There are two G1peaks, diploid and aneuploid (Figure 1). We will allow MultiCycle to fit this fileautomatically. On the [Analyze] menu click the LEFT mouse over the option [AutoFit],hit the F11 key or use the toolbar button . The computer will begin the analysis (thescreen image may change rapidly or flicker as fitting progresses).

Figure 1 Ascii2 data file with two cell cyclesSTEP 3 Observe the fitting results


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The AutoFit result (Figure 2 below) displays the graph and fitting results using two cellcycles and background fitting with aggregation modeling. In addition, [AutoFit ] has

Figure 2 Ascii2 data file after clicking on AutoFit button

performed the [Confidence] analysis of 6 different cell cycle model variations. Thesummary of results from the 6 models, in addition to an overall interpretation of the levelof confidence in the S phase measurement, is also shown.

The aneuploid S phase is 26.6%, with good confidence: the intramodel and intermodel Sphase confidence intervals (the data in parentheses below the %S=26.6 at the lower right)are narrow; the BAD is low (1.2%) and the percent aneuploid cells is adequate. For furtherinformation on the presentation and interpretation of [Confidence] data, see Chapter 11of the MultiCycle for Windows manual.

You may wish to experiment with options listed under the [Options] menu, such as ahigher Y-axis scale using [Options - Rescale X/Y] or the toolbar button . Whenfinished, if you plan to proceed directly to EXAMPLE 2, select the option [Analyze -- Refit ].


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EXAMPLE 2

MANUAL FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOID ANDANEUPLOID) – ASCII.2

If you have just completed an AutoFit analysis of file ASCII.2, and have selected [Refit ],proceed directly to Step 3.

STEP 1 Selecting the File

1. Select the menu option [File – Open ]. The contents of the directory \DEMODATAshould be displayed; if not, change the directory in the usual manner. The file FormatType should be ASCII (If not select this format from the drop-down list). From the listof files in the DATA directory, highlight the file [ASCII.2] by clicking over this namewith the mouse button.

1. The file “ASCII.2” is now colored blue, indicating that it has been chosen. Click themouse over the button labeled [OPEN] in order to begin cell cycle analysis.

STEP 2 Selecting the Analysis Type

1. Since there are two cell cycles in this file (diploid and aneuploid) select the [Analysis]option [2 Cycles] with the mouse or use the toolbar button . The screen will appearas shown in Figure 3 with the diploid G1 having a red line through it & the aneuploidG1, called A1 by MultiCycle having a green line through it. In depth information onwhat two cell cycles are can be found in the other PDF file included with this demo“Introduction to Cell Cycle Analysis:”

Figure 3 Ascii2 data file with two cell cycle option selected

* Note: An Alternate choice is the [Analysis] option [Auto Detect Cycles ]; in this caseMultiCycle will automatically select the two cell cycle option for you. If you use this


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alternative, Step 2 in succeeding examples will also be automatically performed. Note,however, that examples 5 and 6 cannot be performed with [Auto Detect Cycles ]enabled, as these examples use a fitting type other than one, two or three cell cycles.

STEP 3 Fitting Options

Only one fitting option is needed in order to fit this histogram: click on [⌧ BackgroundFit] (see Figure 3) to fit the debris.

STEP 4 Fitting the Background Debris

1. A menu showing all the options for fitting the debris appears as shown below in figure4. To use the default regions and background debris model; simply click on [OK].

Figure 4 Background Debris Fitting Menu of MultiCycle

2. View the fitted background curve (Figure 5). In order to adequately visualize the fit,select a higher Y-axis scale using the option [Options - Rescale X/Y] or press the toolbarbutton three times (8x scale). The display should appear as shown in Figure 5. (Formore information on Background Fitting see “Introduction to Cell Cycle Analysis”).

Figure 5. ASCII.2 withBackground Fit Option executed.


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3. Click on [OK] (Figure 4) to indicate that the Background fitting is satisfactory. Thisbegins the least square analysis.

STEP 5 Begin the least squares analysis.

On the Two Cell Cycle options menu, once the [OK] is clicked the least-squares analysisstarts. As each iteration is completed, the chi-square is reduced. You can observe thisprocess by following the plotted chi-square values if you have a slow computer; otherwisethis screen flashes by to fast to see.

STEP 6 Fitting Results

1. Examine the resulting fitted curves (Figure 6 below). Note that the results display isshown with an 8x expanded Y-axis scale (due to step 4, item 2, above).

Figure 6 Ascii2 data file after manual cell cycle analysisAt this point you can also choose to select [File - Print ] to print to the printer, orexport the data using[File - Export], and selecting from the submenu to save the graphic picture, [Picture ],the numerical data results, [Data ], or both, [All ].

STEP 7 Confidence analysis

1. To begin the analysis of additional models and confidence estimation, click the mousebutton over the [Analyze - Confidence]. In this example, we will choose the model withaggregation modeling added to the sliced nucleus background fitting, so on the[Confidence] submenu, select the option [+Aggregates]. As a more rapid alternative tothe above, use the toolbar button . Fitting of the 5 additional models will begin; as eachof the five least squares analyses is performed, the Chi Square plot will decline (on veryfast computers this may be too rapid to follow).


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2. When fitting of all of the models has been completed, the S and G2 phase confidenceintervals will be shown in the Data section of the display (in parentheses below each Sand G2 estimate on the right of the screen. The intramodel confidence intervals are thetop of each pair of intervals in parentheses, the intermodel intervals are on the bottom- see Chapter 10 of the MultiCycle for Windows manual). If these are not visible, youhave previously selected a simpler Display format (e.g. [Options –Display 3] or[Options – Display 4]); make sure you now select the more compete [Options –Display 1 or Display 2]). Note the 6-model summary table and overall interpretation(Figure 7 below). This display is identical to that obtained by using AutoFit in Example1 (Figure 2), except that we have rescaled the Y-axis by 8x.

Figure 7 Ascii2 data file with complete cell cycle analysis3. When inspection of the results is complete, proceed to the next tutorial example.

Note: It is possible that you may obtain results in this, or other examples, that are not exactly identical to thoseillustrated here. This is especially true if any of the initial estimates have been revised, but occasionallyvariations are also seen between different models of computers. This is because the final results obtained innonlinear least squares analysis depends upon the search for the minimum chi-square, and sometimes thissearch has a slightly different endpoint. The potential variability in this search is described by the intramodelconfidence interval (uppermost of the two ranges in parentheses). Results obtained with different startingconditions will always be within the intermodel confidence interval. It is also possible to refit the data withdifferent fitting options and obtain different analysis results; these results will almost always be withinintermodel confidence interval (lowermost of the two ranges in parentheses).


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EXAMPLE 3

TWO CYCLING POPULATIONS FROM PARAFFIN - FILE ASCII.3

Although similar to Example 2, Example 3 illustrates the nature of a different shape ofdebris that is produced by slicing nuclei in sectioning paraffin blocks. It is the same cellsample as ASCII.2, except that this sample has been paraffin embedded.


1. Select [File - Open ] and follow the directions forStep 1 given in Example 1.

2. Select the file "ASCII.3" This example will showTwo Cell Cycling Populations, Diploid and Aneuploidprepared from paraffin (Figure 8).

3. Click the mouse button on [OK] to begin analysisof the file.

STEP 2 Selecting the Cell Cycle Fit

Choose the [Analyze - 2 Cycles] option with the mouse, or select the toolbar button . If[Auto Detect Cycles] has been previously enabled, this option will have been chosenautomatically.


1. Changes in the Two Cell Cycle fitting options are not needed to get a good fit for thisfile; only background fitting is needed. If you used Example 1 first, the background fit willalready have been automatically performed for this file (Figure 9 on next page) - skip tostep 5.

Figure 8. ASCII.3


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Figure 9 Ascii3 with background debris subtraction

2. If you have not previously fit another file or if you would like to see how to change thedefault settings, select [⌧Background Fit] now to fit the debris.

3. Although the default settings will work, to experiment, select a larger region of thedebris curve to the left of the diploid G1: Click on [Left Region Low] (Figure 10 below)and press the left mouse button when the mouse indicator line is near channel 7; clickon [Left Region High] and press the mouse button when the mouse indicator line isover channel 50. Then click on [OK].

Figure 10 Background Debris Fitting options menu


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4. View the fitted background curve and in the confirmation box (“do you accept thecurrent fit?”), select [Yes].

STEP 4 Begin Least Squares Analysis

In the Two Cell Cycles fitting menu (Figure 9), select [OK] to indicate that you are ready tostart the least-squares analysis.

STEP 5 Observing Fitting Results

Examine the resulting fitted curves (Figure 11 below). Notice the difference from Example2 in the amount of background debris, but the similarity of cell cycle statistics. Thediploid G2 is higher, probably due to a slight skew to the right side of the aneuploid G1peak. Click any mouse button to proceed.

Figure 11 Ascii3 cell cycle analysis

1. To begin the analysis of additional models and confidence estimation, click the mousebutton over the [Analyze - Confidence] option; we will choose the model with aggregationmodeling added to the sliced nucleus background fitting, so click the mouse button overthe option [+ Aggregates]. As a more rapid alternative, use the toolbar button . Fittingof the 5 additional models will begin; as each of the five least squares analyses isperformed, the Chi Square plot will decline. Examine the results of the analysis (Figure12), noting the 6-model summary and overall interpretation, and the S and G2 confidenceintervals in parentheses on the right of the graphic. (Note: if you have previously selecteda simpler Display format [Options -Display 3] or [Options - Display 4], you will need toselect the more compete Display 1 or Display 2 formats at this time, otherwise theSummary and Interpretation will not both be visible). In this example, the 6 fitting modelvariations all yield similar results, the aneuploid cell cycle confidence intervals are narrow(although the diploid cell cycle confidence intervals are wider - the S phase because of the


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abundant sliced nucleus debris overlapping with the diploid S, and the G2 because it ispartially overlapping with the aneuploid G1), the BAD is only moderate and the percentaneuploid cells is good. Thus, the aneuploid S phase confidence is good.

Figure 12 Ascii3 complete cell cycle analysis

2. Choose additional display options. Select [Options - Rescale X/Y] and increase theY-axis scale or press the toolbar button three times.


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EXAMPLE 4

THREE CELL CYCLE POPULATIONS - ASCII.4


Follow the directions for Step 1 given in Example 1, using the [File - Open ] option toselect the file "ASCII.4" (This tutorial example will show three G1 peaks). Click the mousebutton on [OK] to begin analysis of the file


1. Choose the [Analyze - 3 Cycles] option with the mouse, or use the toolbar button .

Note: If you have previously used the option [Auto Detect Cycles ], then the three cellcycle option will have been chosen automatically.


1. Changes in the Three Cell Cycle fitting options are not needed to get a good fit for thisfile. If you used Example 2 or 3 first, the background fit will already be automaticallyperformed for this file (Figure 14).

Note: If you have not previously fit another file, select [Background Fit] now to fit anydebris, then click on [OK] to utilize the default settings, and then click on [Yes].

2. Select [OK] to indicate that you are ready to start the least-squares analysis


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STEP 4 Fitting Results and Confidence Analysis

Figure 15 Ascii4 preliminary cell cycle analysis

1. Examine the initial fitting results (Figure 15). Then select the menu option [Analyze -Confidence]; for this example, we will choose aggregate modeling added to the slicednucleus background fitting; therefore click the LEFT mouse button over the option[+Aggregates]. As a more rapid alternative to using the menu, use the toolbar button

.

2. Fitting of the 5 additional models will begin; as each of the five least squares analysesis performed, the Chi-Square plot will decline (on very fast computers this process maybe too rapid to visualize).

Examine the results of the [Confidence] analysis, noting the 6-model summary andoverall interpretation below the graph, and the S and G2 confidence intervals inparentheses on the right of the screen (Figure 16).


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Figure 16 Ascii4 complete cell cycle analysis

In this example, the 6 fitting model variations all yield approximately similar results,except for the diploid S, for which the intramodel confidence interval also is wide,consistent with poor confidence (presumably due to the extensive sliced nucleus debrisoverlapping the diploid S). The aneuploid cell cycle confidence intervals are reasonablynarrow. The BAD (except over the diploid cell cycle, note Diploid BAD) is moderately lowand the percent aneuploid cells are adequate. Thus, the confidence for both aneuploid Sphases is good.

2. You may choose to print the results ([File - Print ]), or perhaps refit the data([Analysis -- Refit ]).


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EXAMPLE 5

ONE CELL CYCLE AND ONE ADDITIONAL PEAK - ASCII.1

This file is most optimally fit as two cell cycles, however, for purposes of this illustration,we will demonstrate fitting with one cell cycle and one additional G1 peak fit with the[Overlapped Peak] option.


1. Follow the directions for Step 1 given in Example 1 to select ASCII file types, and usethe option [File-Open ] to select the file.

2. Click the mouse over the file "ASCII.1".

3. Click the mouse button on [OK] to begin analysis of the file.


1. Choose the [Analyze – One Cycle + Overlapped Peak] menu option with the mouse.

Note: If you have previously selected the [Auto Detect Cycles ] option, MultiCycle willhave automatically selected the Two Cell Cycle fitting model. In this case, select[Cancel] from the Two Cell Cycle fitting option menu, and then select [Analyze - OneCycle + Overlapped Peak].

2. On the submenu that appears, click on [Diploid] to indicate that the peak is a diploidcell population. This selection is based on prior knowledge (e.g. from a referencestandard).

Figure 17. One Cell Cycle and OneAdditional Peak


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* Note: 95% of all diploid peaks will be found to the left of an aneuploid peak. This isbecause the great majority of aneuploid cells have hyper-(greater than) diploid DNAcontent.

STEP 3 Selecting Fitting Options

1. On the Added Diploid Peak fitting options menu (Figure 18 below), click on the [Orderof S] option [2] in order to select a second order (curved) polynomial S-phase shape.

2. If the background debris has already been automatically performed ([⌧ BackgroundFit] checked), proceed to step 5; otherwise; select the [ Background Fit] option to fit thedebris.

STEP 4 Fitting the Background Debris

1. To use the default regions and fitting model; simply click on [OK].

2. View the fitted background curve and click on [Yes] when asked if you accept the fit.This returns you to the fitting options page.

Figure 18 Ascii1 diploid peak with aneuploid cell cycle

* Note: For more information on Background Fitting see the “Introduction to Cell CycleAnalysis PDF file.


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STEP 5 Beginning the Least Squares Analysis

On the Overlapped Peak fitting options menu (Figure 19), select [OK] to indicate that youare ready to start the least-squares analysis. As it is completed, the chi-square is

Figure 19 Ascii1 completed cell cycle analysis

improved (reduced). You can observe this process by following the plotted chi-squarevalues.

STEP 6 Finished Results

1. Examine your results (Figure 19). Choose the [Options - Rescale X/Y] option, andselect a Y-axis scaling of 4, or instead, press the toolbar button twice. At this point youcan print the results, proceed to the next file, select different [Options - Display] options,or refit the data (for example, refit this histogram with the two cell cycle fitting option).


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EXAMPLE 6

SYNCHRONIZED CELLS IN S-PHASE - ASCII.5

This option will be of use in fitting populations of cells with a perturbed cell cycle, as fromin vitro synchronization by cell cycle blocking agents.


Follow the directions for Step 1 given in Example 1 to select the file "ASCII.5 using theoption [File-Open ] menu option. Select [OK] to begin analysis of the file.

STEP 2. Selecting the Cell Cycle Fit

Note: If you have previously used the option [Auto Detect Cycles ], then the Two CellCycle analysis menu will have been automatically, and incorrectly, chosen. You willneed to exit this menu by selecting [Cancel] on the two cycle fitting menu, then proceedas described below.

1. Choose the [Analyze - Synchronous S-phase] option.

1. The software will automatically place a cursor over the peak in the middle of the cellcycle data - this is the excess S-phase that would not be satisfactorily fit when usingthe usual mode. In this example the peak is near mid-S-phase.

Figure 20 Ascii5 with Synchronous S phase fitting options


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MultiCycle will display its estimates of the G1 and G2 mean positions (see Figure 20]. Youcan improve the speed of the fitting process by improving the G2 position estimate: Clickon [G2 Mean] and click again when the mouse cursor is over channel ~120. Note that thefitting process can also manage with the default selection. Also note that the option [G2CV = G1 CV] has been automatically selected for you. This is often a very useful optionwhen analyzing synchronized cells, because there are many cases where either the G1 orthe G2 is not distinctly separated from the S phase cells. The computer estimate of thepeak of the S phase population is also shown. This should be examined, however, theestimate is satisfactory in this case.

Click on [OK] to indicate that you are ready to start the least-squares analysis.

STEP 4 Final Results - Data Display Options

Figure 21 Ascii5 completed cell cycle analysis

Examine the fitting results (Figure 21). Note that the cell cycle data includes a descriptionof the mean channel of the total S phase, 83.3; this information can be used to calculatethe information that the average S phase cell has completed 41% of S phase: the G2-G1interval is 63 channels, the mean S phase is 83.2-57.8=25.4 channels above the G1, 100x 25.4/63 = 42%. There are also statistics for the portion of the S phase that appears tobe synchronized: it is centered over a mean channel of 80.1, and the synchronized portionrepresents 65.7% of all cells.

Examine the [Options] menu. It is useful to select [Rescale X/Y ], etc. Choose to printthe graphic results with [File - Print ], proceed to the next file, and view the data in thesimpler [Options - Display 3 or Display 4] formats (see, or perhaps refit the data.


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Figure 21 Ascii5 with simplified data display

If you decide to refit the data, experiment with the results of allowing the G2 CV to bedifferent from that of the G1 CV by unchecking the option [G2 CV = G1 CV].

This concludes our tutorial. Hopefully, by now, you have a feel for the ease of use of theMultiCycle for Windows software. Remember that cell cycle analysis software is only atool, the human operator must perform the final analysis.

If you have a question, do not hesitate to contact us at [email protected]

Thank you.

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multicycle for windows tutorial