Multicycle For Windows Tutorial
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MultiCycle for Windows TUTORIAL Provided by
Phoenix Flow Systems, Inc. 6790 Top Gun St. #1 San Diego, CA 92121 USA 858 453-5095 fax 858 453-2117 www.phoenixflow.com Tutorial Introduction
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Example 1 - AutoFit analysis of ASCII.2 Automatic Fitting of a Two Cell Cycle Histogram
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Example 2 - ASCII.2 Manual Fitting of a Two Cell Cycle Histogram
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Example 3 - ASCII.3 Manual Fitting of Two Cell Cycle Populations from Paraffin
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Example 4 - ASCII.4 Manual Fitting of Three Cell Cycle Populations (One Diploid and Two Aneuploid Peaks)
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Example 5 - ASCII.1 One Cell Cycle and One DNA Peak
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Example 6 - ASCII.5 Synchronized Cultured Cells in S Phase
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1 Phoenix Flow Systems, Inc. www.phoenixflow.com
TUTORIAL INTRODUCTION In this tutorial you will learn how to use MultiCycle to analyze different cell cycle histogram types and file formats. There are six different tutorial examples using files included with MultiCycle for Windows (located in the DEMODATA directory) that will illustrate the use of MultiCycle. If you have no knowledge of performing a DNA cell cycle analysis or need a refresher, we strongly recommend you read the other PDF file installed with the MultiCycle for Windows, “Introduction to Cell Cycle Analysis.” DNA cell cycle analysis does not have to be difficult. Interpreting the data is the hard part. Cell cycle analysis with MultiCycle for Windows is performed with these steps: 1. 2. 3. 4. 5.
Select a data file Determine the number of total cell cycles in the histogram Subtract the debris from the histogram Perform the analysis Evaluate the results
MultiCycle for Windows has a menu bar right below the main menu, which leads you through the above process of performing a cell cycle analysis. Only the appropriate 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 histogram This 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. 2 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 1 AUTOMATIC FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOID AND 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 the directory DEMODATA should be displayed; if not, change the directory in the usual Windows manner looking for \Program Files\Phoenix Flow Systems\MultiCycle for Windows\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 open menu box.
2. From the list of files within this directory, highlight the file [ASCII.2] by clicking over this name with the mouse button. The file "ASCII.2" is now colored blue, indicating that it has been chosen. 3. Click the mouse button on [Open] to begin analysis of this file.
3 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 G1 peaks, diploid and aneuploid (Figure 1). We will allow MultiCycle to fit this file automatically. On the [Analyze] menu click the LEFT mouse over the option [AutoFit], . The computer will begin the analysis (the hit the F11 key or use the toolbar button screen image may change rapidly or flicker as fitting progresses).
STEP 3
Figure 1 Ascii2 data file with two cell cycles Observe the fitting results
4 Phoenix Flow Systems, Inc. www.phoenixflow.com
The AutoFit result (Figure 2 below) displays the graph and fitting results using two cell ] has cycles and background fitting with aggregation modeling. In addition, [AutoFit
Figure 2 Ascii2 data file after clicking on AutoFit button
performed the [Confidence] analysis of 6 different cell cycle model variations. The summary of results from the 6 models, in addition to an overall interpretation of the level of confidence in the S phase measurement, is also shown. The aneuploid S phase is 26.6%, with good confidence: the intramodel and intermodel S phase 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 further information on the presentation and interpretation of [Confidence] data, see Chapter 11 of the MultiCycle for Windows manual. You may wish to experiment with options listed under the [Options] menu, such as a . When higher Y-axis scale using [Options - Rescale X/Y] or the toolbar button finished, if you plan to proceed directly to EXAMPLE 2, select the option [Analyze -- Refit ].
5 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 2 MANUAL FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOID AND ANEUPLOID) – 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 \DEMODATA should be displayed; if not, change the directory in the usual manner. The file Format Type should be ASCII (If not select this format from the drop-down list). From the list of files in the DATA directory, highlight the file [ASCII.2] by clicking over this name with the mouse button. 1. The file “ASCII.2” is now colored blue, indicating that it has been chosen. Click the mouse 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] . The screen will appear option [2 Cycles] with the mouse or use the toolbar button as shown in Figure 3 with the diploid G1 having a red line through it & the aneuploid G1, called A1 by MultiCycle having a green line through it. In depth information on what 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 case MultiCycle will automatically select the two cell cycle option for you. If you use this 6 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 [⌧ Background Fit] (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 figure 4. 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 toolbar three times (8x scale). The display should appear as shown in Figure 5. (For button more information on Background Fitting see “Introduction to Cell Cycle Analysis”).
Figure 5. ASCII.2 with Background Fit Option executed.
7 Phoenix Flow Systems, Inc. www.phoenixflow.com
3. Click on [OK] (Figure 4) to indicate that the Background fitting is satisfactory. This begins 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 analysis starts. As each iteration is completed, the chi-square is reduced. You can observe this process by following the plotted chi-square values if you have a slow computer; otherwise this 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 is shown with an 8x expanded Y-axis scale (due to step 4, item 2, above).
Figure 6 Ascii2 data file after manual cell cycle analysis ] to print to the printer, or At this point you can also choose to select [File - Print export 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 mouse button over the [Analyze - Confidence]. In this example, we will choose the model with aggregation modeling added to the sliced nucleus background fitting, so on the [Confidence] submenu, select the option [+Aggregates]. As a more rapid alternative to the above, use the toolbar button . Fitting of the 5 additional models will begin; as each of the five least squares analyses is performed, the Chi Square plot will decline (on very fast computers this may be too rapid to follow). 8 Phoenix Flow Systems, Inc. www.phoenixflow.com
2. When fitting of all of the models has been completed, the S and G2 phase confidence intervals will be shown in the Data section of the display (in parentheses below each S and G2 estimate on the right of the screen. The intramodel confidence intervals are the top 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, you have 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 Example 1 (Figure 2), except that we have rescaled the Y-axis by 8x.
Figure 7 Ascii2 data file with complete cell cycle analysis 3. 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 those illustrated here. This is especially true if any of the initial estimates have been revised, but occasionally variations are also seen between different models of computers. This is because the final results obtained in nonlinear least squares analysis depends upon the search for the minimum chi-square, and sometimes this search has a slightly different endpoint. The potential variability in this search is described by the intramodel confidence interval (uppermost of the two ranges in parentheses). Results obtained with different starting conditions will always be within the intermodel confidence interval. It is also possible to refit the data with different fitting options and obtain different analysis results; these results will almost always be within intermodel confidence interval (lowermost of the two ranges in parentheses).
9 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 3 TWO CYCLING POPULATIONS FROM PARAFFIN - FILE ASCII.3 Although similar to Example 2, Example 3 illustrates the nature of a different shape of debris that is produced by slicing nuclei in sectioning paraffin blocks. It is the same cell sample as ASCII.2, except that this sample has been paraffin embedded. STEP 1
Selecting the File ] and follow the directions for 1. Select [File - Open Step 1 given in Example 1. 2. Select the file "ASCII.3" This example will show Two Cell Cycling Populations, Diploid and Aneuploid prepared from paraffin (Figure 8). 3. Click the mouse button on [OK] to begin analysis of the file.
Figure 8. ASCII.3
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 chosen automatically. STEP 3
Fitting Options
1. Changes in the Two Cell Cycle fitting options are not needed to get a good fit for this file; only background fitting is needed. If you used Example 1 first, the background fit will already have been automatically performed for this file (Figure 9 on next page) - skip to step 5.
10 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 the default settings, select [⌧Background Fit] now to fit the debris. 3. Although the default settings will work, to experiment, select a larger region of the debris 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; click on [Left Region High] and press the mouse button when the mouse indicator line is over channel 50. Then click on [OK].
Figure 10 Background Debris Fitting options menu
11 Phoenix Flow Systems, Inc. www.phoenixflow.com
4. View the fitted background curve and in the confirmation box (“do you accept the current 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 to start the least-squares analysis. STEP 5
Observing Fitting Results
Examine the resulting fitted curves (Figure 11 below). Notice the difference from Example 2 in the amount of background debris, but the similarity of cell cycle statistics. The diploid G2 is higher, probably due to a slight skew to the right side of the aneuploid G1 peak. 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 mouse button over the [Analyze - Confidence] option; we will choose the model with aggregation modeling added to the sliced nucleus background fitting, so click the mouse button over the option [+ Aggregates]. As a more rapid alternative, use the toolbar button . Fitting of the 5 additional models will begin; as each of the five least squares analyses is performed, the Chi Square plot will decline. Examine the results of the analysis (Figure 12), noting the 6-model summary and overall interpretation, and the S and G2 confidence intervals in parentheses on the right of the graphic. (Note: if you have previously selected a simpler Display format [Options -Display 3] or [Options - Display 4], you will need to select the more compete Display 1 or Display 2 formats at this time, otherwise the Summary and Interpretation will not both be visible). In this example, the 6 fitting model variations 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 12 Phoenix Flow Systems, Inc. www.phoenixflow.com
abundant sliced nucleus debris overlapping with the diploid S, and the G2 because it is partially overlapping with the aneuploid G1), the BAD is only moderate and the percent aneuploid 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 the Y-axis scale or press the toolbar button three times.
13 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 4 THREE CELL CYCLE POPULATIONS STEP 1
ASCII.4
Selecting the File
] option to Follow the directions for Step 1 given in Example 1, using the [File - Open select the file "ASCII.4" (This tutorial example will show three G1 peaks). Click the mouse button on [OK] to begin analysis of the file STEP 2
Selecting the Cell Cycle Fit
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 cycle option will have been chosen automatically. STEP 3
.
], then the three cell
Fitting Options
1. Changes in the Three Cell Cycle fitting options are not needed to get a good fit for this file. If you used Example 2 or 3 first, the background fit will already be automatically performed for this file (Figure 14). Note: If you have not previously fit another file, select [Background Fit] now to fit any debris, 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
14 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 sliced nucleus 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 analyses is performed, the Chi-Square plot will decline (on very fast computers this process may be too rapid to visualize). Examine the results of the [Confidence] analysis, noting the 6-model summary and overall interpretation below the graph, and the S and G2 confidence intervals in parentheses on the right of the screen (Figure 16). 15 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 debris overlapping the diploid S). The aneuploid cell cycle confidence intervals are reasonably narrow. The BAD (except over the diploid cell cycle, note Diploid BAD) is moderately low and the percent aneuploid cells are adequate. Thus, the confidence for both aneuploid S phases is good. 2. You may choose to print the results ([File - Print ]). ([Analysis -- Refit
16 Phoenix Flow Systems, Inc. www.phoenixflow.com
]), or perhaps refit the data
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. STEP 1
Selecting the File
1. Follow the directions for Step 1 given in Example 1 to select ASCII file types, and use the option [File-Open ] to select the file. 2. Click the mouse over the file "ASCII.1".
Figure 17. One Cell Cycle and One Additional Peak
3. Click the mouse button on [OK] to begin analysis of the file. STEP 2
Selecting the Cell Cycle Fit
1. Choose the [Analyze – One Cycle + Overlapped Peak] menu option with the mouse. ] option, MultiCycle will Note: If you have previously selected the [Auto Detect Cycles have 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 - One Cycle + Overlapped Peak]. 2. On the submenu that appears, click on [Diploid] to indicate that the peak is a diploid cell population. This selection is based on prior knowledge (e.g. from a reference standard).
17 Phoenix Flow Systems, Inc. www.phoenixflow.com
* Note: 95% of all diploid peaks will be found to the left of an aneuploid peak. This is because the great majority of aneuploid cells have hyper-(greater than) diploid DNA content. STEP 3
Selecting Fitting Options
1. On the Added Diploid Peak fitting options menu (Figure 18 below), click on the [Order of 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 ([⌧ Background Fit] checked), proceed to step 5; otherwise; select the [ Background Fit] option to fit the debris. 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 Cycle Analysis PDF file.
18 Phoenix Flow Systems, Inc. www.phoenixflow.com
STEP 5
Beginning the Least Squares Analysis
On the Overlapped Peak fitting options menu (Figure 19), select [OK] to indicate that you are ready to start the least-squares analysis. As it is completed, the chi-square is
Figure 19 Ascii1 completed cell cycle analysis improved (reduced). values. STEP 6
You can observe this process by following the plotted chi-square
Finished Results
1. Examine your results (Figure 19). Choose the [Options - Rescale X/Y] option, and select a Y-axis scaling of 4, or instead, press the toolbar button twice. At this point you can 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).
19 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 from in vitro synchronization by cell cycle blocking agents. STEP 1
Selecting the File
Follow the directions for Step 1 given in Example 1 to select the file "ASCII.5 using the option [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 Cell Cycle analysis menu will have been automatically, and incorrectly, chosen. You will need to exit this menu by selecting [Cancel] on the two cycle fitting menu, then proceed as 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 cell cycle data - this is the excess S-phase that would not be satisfactorily fit when using the usual mode. In this example the peak is near mid-S-phase.
Figure 20 Ascii5 with Synchronous S phase fitting options
20 Phoenix Flow Systems, Inc. www.phoenixflow.com
STEP 3
Fitting Options
MultiCycle will display its estimates of the G1 and G2 mean positions (see Figure 20]. You can improve the speed of the fitting process by improving the G2 position estimate: Click on [G2 Mean] and click again when the mouse cursor is over channel ~120. Note that the fitting process can also manage with the default selection. Also note that the option [G2 CV = G1 CV] has been automatically selected for you. This is often a very useful option when analyzing synchronized cells, because there are many cases where either the G1 or the G2 is not distinctly separated from the S phase cells. The computer estimate of the peak of the S phase population is also shown. This should be examined, however, the estimate 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 description of the mean channel of the total S phase, 83.3; this information can be used to calculate the information that the average S phase cell has completed 41% of S phase: the G2-G1 interval is 63 channels, the mean S phase is 83.2-57.8=25.4 channels above the G1, 100 x 25.4/63 = 42%. There are also statistics for the portion of the S phase that appears to be synchronized: it is centered over a mean channel of 80.1, and the synchronized portion represents 65.7% of all cells. Examine the [Options] menu. It is useful to select [Rescale X/Y ], etc. Choose to print ], proceed to the next file, and view the data in the the graphic results with [File - Print simpler [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 be different 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 the MultiCycle for Windows software. Remember that cell cycle analysis software is only a tool, the human operator must perform the final analysis. If you have a question, do not hesitate to contact us at [email protected] Thank you.
22 Phoenix Flow Systems, Inc. www.phoenixflow.com
Phoenix Flow Systems, Inc. 6790 Top Gun St. #1 San Diego, CA 92121 USA 858 453-5095 fax 858 453-2117 www.phoenixflow.com Tutorial Introduction
2
Example 1 - AutoFit analysis of ASCII.2 Automatic Fitting of a Two Cell Cycle Histogram
3
Example 2 - ASCII.2 Manual Fitting of a Two Cell Cycle Histogram
6
Example 3 - ASCII.3 Manual Fitting of Two Cell Cycle Populations from Paraffin
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Example 4 - ASCII.4 Manual Fitting of Three Cell Cycle Populations (One Diploid and Two Aneuploid Peaks)
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Example 5 - ASCII.1 One Cell Cycle and One DNA Peak
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Example 6 - ASCII.5 Synchronized Cultured Cells in S Phase
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1 Phoenix Flow Systems, Inc. www.phoenixflow.com
TUTORIAL INTRODUCTION In this tutorial you will learn how to use MultiCycle to analyze different cell cycle histogram types and file formats. There are six different tutorial examples using files included with MultiCycle for Windows (located in the DEMODATA directory) that will illustrate the use of MultiCycle. If you have no knowledge of performing a DNA cell cycle analysis or need a refresher, we strongly recommend you read the other PDF file installed with the MultiCycle for Windows, “Introduction to Cell Cycle Analysis.” DNA cell cycle analysis does not have to be difficult. Interpreting the data is the hard part. Cell cycle analysis with MultiCycle for Windows is performed with these steps: 1. 2. 3. 4. 5.
Select a data file Determine the number of total cell cycles in the histogram Subtract the debris from the histogram Perform the analysis Evaluate the results
MultiCycle for Windows has a menu bar right below the main menu, which leads you through the above process of performing a cell cycle analysis. Only the appropriate 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 histogram This 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. 2 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 1 AUTOMATIC FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOID AND 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 the directory DEMODATA should be displayed; if not, change the directory in the usual Windows manner looking for \Program Files\Phoenix Flow Systems\MultiCycle for Windows\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 open menu box.
2. From the list of files within this directory, highlight the file [ASCII.2] by clicking over this name with the mouse button. The file "ASCII.2" is now colored blue, indicating that it has been chosen. 3. Click the mouse button on [Open] to begin analysis of this file.
3 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 G1 peaks, diploid and aneuploid (Figure 1). We will allow MultiCycle to fit this file automatically. On the [Analyze] menu click the LEFT mouse over the option [AutoFit], . The computer will begin the analysis (the hit the F11 key or use the toolbar button screen image may change rapidly or flicker as fitting progresses).
STEP 3
Figure 1 Ascii2 data file with two cell cycles Observe the fitting results
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The AutoFit result (Figure 2 below) displays the graph and fitting results using two cell ] has cycles and background fitting with aggregation modeling. In addition, [AutoFit
Figure 2 Ascii2 data file after clicking on AutoFit button
performed the [Confidence] analysis of 6 different cell cycle model variations. The summary of results from the 6 models, in addition to an overall interpretation of the level of confidence in the S phase measurement, is also shown. The aneuploid S phase is 26.6%, with good confidence: the intramodel and intermodel S phase 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 further information on the presentation and interpretation of [Confidence] data, see Chapter 11 of the MultiCycle for Windows manual. You may wish to experiment with options listed under the [Options] menu, such as a . When higher Y-axis scale using [Options - Rescale X/Y] or the toolbar button finished, if you plan to proceed directly to EXAMPLE 2, select the option [Analyze -- Refit ].
5 Phoenix Flow Systems, Inc. www.phoenixflow.com
EXAMPLE 2 MANUAL FITTING OF TWO CELL CYCLE POPULATIONS (DIPLOID AND ANEUPLOID) – 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 \DEMODATA should be displayed; if not, change the directory in the usual manner. The file Format Type should be ASCII (If not select this format from the drop-down list). From the list of files in the DATA directory, highlight the file [ASCII.2] by clicking over this name with the mouse button. 1. The file “ASCII.2” is now colored blue, indicating that it has been chosen. Click the mouse 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] . The screen will appear option [2 Cycles] with the mouse or use the toolbar button as shown in Figure 3 with the diploid G1 having a red line through it & the aneuploid G1, called A1 by MultiCycle having a green line through it. In depth information on what 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 case MultiCycle will automatically select the two cell cycle option for you. If you use this 6 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 [⌧ Background Fit] (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 figure 4. 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 toolbar three times (8x scale). The display should appear as shown in Figure 5. (For button more information on Background Fitting see “Introduction to Cell Cycle Analysis”).
Figure 5. ASCII.2 with Background Fit Option executed.
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3. Click on [OK] (Figure 4) to indicate that the Background fitting is satisfactory. This begins 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 analysis starts. As each iteration is completed, the chi-square is reduced. You can observe this process by following the plotted chi-square values if you have a slow computer; otherwise this 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 is shown with an 8x expanded Y-axis scale (due to step 4, item 2, above).
Figure 6 Ascii2 data file after manual cell cycle analysis ] to print to the printer, or At this point you can also choose to select [File - Print export 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 mouse button over the [Analyze - Confidence]. In this example, we will choose the model with aggregation modeling added to the sliced nucleus background fitting, so on the [Confidence] submenu, select the option [+Aggregates]. As a more rapid alternative to the above, use the toolbar button . Fitting of the 5 additional models will begin; as each of the five least squares analyses is performed, the Chi Square plot will decline (on very fast computers this may be too rapid to follow). 8 Phoenix Flow Systems, Inc. www.phoenixflow.com
2. When fitting of all of the models has been completed, the S and G2 phase confidence intervals will be shown in the Data section of the display (in parentheses below each S and G2 estimate on the right of the screen. The intramodel confidence intervals are the top 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, you have 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 Example 1 (Figure 2), except that we have rescaled the Y-axis by 8x.
Figure 7 Ascii2 data file with complete cell cycle analysis 3. 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 those illustrated here. This is especially true if any of the initial estimates have been revised, but occasionally variations are also seen between different models of computers. This is because the final results obtained in nonlinear least squares analysis depends upon the search for the minimum chi-square, and sometimes this search has a slightly different endpoint. The potential variability in this search is described by the intramodel confidence interval (uppermost of the two ranges in parentheses). Results obtained with different starting conditions will always be within the intermodel confidence interval. It is also possible to refit the data with different fitting options and obtain different analysis results; these results will almost always be within intermodel 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 of debris that is produced by slicing nuclei in sectioning paraffin blocks. It is the same cell sample as ASCII.2, except that this sample has been paraffin embedded. STEP 1
Selecting the File ] and follow the directions for 1. Select [File - Open Step 1 given in Example 1. 2. Select the file "ASCII.3" This example will show Two Cell Cycling Populations, Diploid and Aneuploid prepared from paraffin (Figure 8). 3. Click the mouse button on [OK] to begin analysis of the file.
Figure 8. ASCII.3
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 chosen automatically. STEP 3
Fitting Options
1. Changes in the Two Cell Cycle fitting options are not needed to get a good fit for this file; only background fitting is needed. If you used Example 1 first, the background fit will already have been automatically performed for this file (Figure 9 on next page) - skip to step 5.
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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 the default settings, select [⌧Background Fit] now to fit the debris. 3. Although the default settings will work, to experiment, select a larger region of the debris 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; click on [Left Region High] and press the mouse button when the mouse indicator line is over 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 the current 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 to start the least-squares analysis. STEP 5
Observing Fitting Results
Examine the resulting fitted curves (Figure 11 below). Notice the difference from Example 2 in the amount of background debris, but the similarity of cell cycle statistics. The diploid G2 is higher, probably due to a slight skew to the right side of the aneuploid G1 peak. 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 mouse button over the [Analyze - Confidence] option; we will choose the model with aggregation modeling added to the sliced nucleus background fitting, so click the mouse button over the option [+ Aggregates]. As a more rapid alternative, use the toolbar button . Fitting of the 5 additional models will begin; as each of the five least squares analyses is performed, the Chi Square plot will decline. Examine the results of the analysis (Figure 12), noting the 6-model summary and overall interpretation, and the S and G2 confidence intervals in parentheses on the right of the graphic. (Note: if you have previously selected a simpler Display format [Options -Display 3] or [Options - Display 4], you will need to select the more compete Display 1 or Display 2 formats at this time, otherwise the Summary and Interpretation will not both be visible). In this example, the 6 fitting model variations 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 12 Phoenix Flow Systems, Inc. www.phoenixflow.com
abundant sliced nucleus debris overlapping with the diploid S, and the G2 because it is partially overlapping with the aneuploid G1), the BAD is only moderate and the percent aneuploid 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 the Y-axis scale or press the toolbar button three times.
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EXAMPLE 4 THREE CELL CYCLE POPULATIONS STEP 1
ASCII.4
Selecting the File
] option to Follow the directions for Step 1 given in Example 1, using the [File - Open select the file "ASCII.4" (This tutorial example will show three G1 peaks). Click the mouse button on [OK] to begin analysis of the file STEP 2
Selecting the Cell Cycle Fit
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 cycle option will have been chosen automatically. STEP 3
.
], then the three cell
Fitting Options
1. Changes in the Three Cell Cycle fitting options are not needed to get a good fit for this file. If you used Example 2 or 3 first, the background fit will already be automatically performed for this file (Figure 14). Note: If you have not previously fit another file, select [Background Fit] now to fit any debris, 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 sliced nucleus 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 analyses is performed, the Chi-Square plot will decline (on very fast computers this process may be too rapid to visualize). Examine the results of the [Confidence] analysis, noting the 6-model summary and overall interpretation below the graph, and the S and G2 confidence intervals in parentheses on the right of the screen (Figure 16). 15 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 debris overlapping the diploid S). The aneuploid cell cycle confidence intervals are reasonably narrow. The BAD (except over the diploid cell cycle, note Diploid BAD) is moderately low and the percent aneuploid cells are adequate. Thus, the confidence for both aneuploid S phases is good. 2. You may choose to print the results ([File - Print ]). ([Analysis -- Refit
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]), or perhaps refit the data
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. STEP 1
Selecting the File
1. Follow the directions for Step 1 given in Example 1 to select ASCII file types, and use the option [File-Open ] to select the file. 2. Click the mouse over the file "ASCII.1".
Figure 17. One Cell Cycle and One Additional Peak
3. Click the mouse button on [OK] to begin analysis of the file. STEP 2
Selecting the Cell Cycle Fit
1. Choose the [Analyze – One Cycle + Overlapped Peak] menu option with the mouse. ] option, MultiCycle will Note: If you have previously selected the [Auto Detect Cycles have 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 - One Cycle + Overlapped Peak]. 2. On the submenu that appears, click on [Diploid] to indicate that the peak is a diploid cell population. This selection is based on prior knowledge (e.g. from a reference standard).
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* Note: 95% of all diploid peaks will be found to the left of an aneuploid peak. This is because the great majority of aneuploid cells have hyper-(greater than) diploid DNA content. STEP 3
Selecting Fitting Options
1. On the Added Diploid Peak fitting options menu (Figure 18 below), click on the [Order of 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 ([⌧ Background Fit] checked), proceed to step 5; otherwise; select the [ Background Fit] option to fit the debris. 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 Cycle Analysis 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 you are ready to start the least-squares analysis. As it is completed, the chi-square is
Figure 19 Ascii1 completed cell cycle analysis improved (reduced). values. STEP 6
You can observe this process by following the plotted chi-square
Finished Results
1. Examine your results (Figure 19). Choose the [Options - Rescale X/Y] option, and select a Y-axis scaling of 4, or instead, press the toolbar button twice. At this point you can 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).
19 Phoenix Flow Systems, Inc. www.phoenixflow.com
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 from in vitro synchronization by cell cycle blocking agents. STEP 1
Selecting the File
Follow the directions for Step 1 given in Example 1 to select the file "ASCII.5 using the option [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 Cell Cycle analysis menu will have been automatically, and incorrectly, chosen. You will need to exit this menu by selecting [Cancel] on the two cycle fitting menu, then proceed as 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 cell cycle data - this is the excess S-phase that would not be satisfactorily fit when using the 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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STEP 3
Fitting Options
MultiCycle will display its estimates of the G1 and G2 mean positions (see Figure 20]. You can improve the speed of the fitting process by improving the G2 position estimate: Click on [G2 Mean] and click again when the mouse cursor is over channel ~120. Note that the fitting process can also manage with the default selection. Also note that the option [G2 CV = G1 CV] has been automatically selected for you. This is often a very useful option when analyzing synchronized cells, because there are many cases where either the G1 or the G2 is not distinctly separated from the S phase cells. The computer estimate of the peak of the S phase population is also shown. This should be examined, however, the estimate 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 description of the mean channel of the total S phase, 83.3; this information can be used to calculate the information that the average S phase cell has completed 41% of S phase: the G2-G1 interval is 63 channels, the mean S phase is 83.2-57.8=25.4 channels above the G1, 100 x 25.4/63 = 42%. There are also statistics for the portion of the S phase that appears to be synchronized: it is centered over a mean channel of 80.1, and the synchronized portion represents 65.7% of all cells. Examine the [Options] menu. It is useful to select [Rescale X/Y ], etc. Choose to print ], proceed to the next file, and view the data in the the graphic results with [File - Print simpler [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 be different 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 the MultiCycle for Windows software. Remember that cell cycle analysis software is only a tool, 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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