Cell Cycle
FlowJo provides a simple interface to performing fairly sophisticated DNA/Cell Cycle analysis. To launch the Cell Cycle platform, select any sample or gated population (i.e., where you have gated out debris or gated for a desired phenotype), and choose Cell Cycle... from the Workspace menu. FlowJo brings up a graph window that is specially designed for cell cycle analyses. You might want to visit the page giving hints for Cell Cycle analysis with FlowJo.
FlowJo tries to determine which parameter contains the DNA quantitation information; if it chooses the wrong one, select the correct one from the X-axis popup menu. Then click on the button "Add/Change Models" to begin your Cell Cycle analysis.
When you click on the Models disclosure triangle , FlowJo shows you the options below. From this area, you can choose between the Watson (Pragmatic) and the Dean-Jett-Fox models to compute for the data, and, if necessary, to constrain the fitting parameters by any number of criteria. The Watson model makes no assumptions about the shape of the S-Phase distribution; it (by definition) fits the S-phase exactly. The DJF model assumes that the S-phase is can be modeled by a second degree polynomial (that is convoluted with gaussian distributions of varying width throughout S-phase). You can also choose to have a synchronized peak within the S-phase as an option to this model.
Once FlowJo has computed the model, it displays the fit along with statistical data in the Cell Cycle window, such as that shown below.
The statistics displayed in a column on the graph will depend on which model and options are computed, but both will include basic statistics such as the fraction of cells in G1, S, and G2, the positions of the G1 and G2 peaks (and their widths), and the number of cells below G1 and above G2. In addition, the RMS (root mean square) error of the fit is displayed at the top of the list. If you change the fitting criteria, you may wish to minimize this value as a way to optimize the fit. If FlowJo fails to fit the model(s) to the data, then it will display "Invalid" in the RMS field. In this case, you will want to help FlowJo fit the data by constraining different parameters. See the Hints page for ideas on how to proceed.
Choosing Split S Phase Stats will add percent values for S1, S2, S3 and S4 to the values listed in the column on the graph. Experiment by checking the boxes that Draw Model Sum, Draw Components and Use Pattern Fill to see if they display useful detail in your analysis. The color boxes let you choose what colors will be displayed for the given parameter.
In the Constraints area , the fit was constrained such that the G2 peak must be found within the defined range. Checking Edit G1/G2 Curves produces vertical lines marked G1 and G2 on the graph. You can drag these lines to visually adjust these gates to the graph. Dragging vertically will include a wider range of values from the x axis, dragging horizontally will move the peak of the gate along the histogram line.
At the top of this window is the statistics button. By clicking it, you can choose to add any of the listed statistics to the display. I have chosen Median FL2-Area and it is displayed as the red line on the graph.
Selecting the Create Gates button adds gates discribing the non-overlapping areas of the cell cycle populations to the selected parent population in the Workspace. Cell cycle analyses can be copied between subsets and between samples, and even to groups, just like every other analysis in FlowJo. In this fashion, you can compute Cell Cycle analyses on every sample in an experiment. In general, you will begin by analyzing a control sample, and use this control sample to define ranges for G1 and G2. If you have unusual distributions, constraining the fit by these ranges will help FlowJo determine the proper distribution of cells. Once you have defined the ranges and the fit, drag the analysis to other samples (or the group).
Selecting the Show Overlay button produces a histogram overlay in the Layout Editor showing the three subpopulations overlaid on the same graph space.
You can drag Cell Cycle analyses from the Workspace to the Layout Editor to generate reports that contain the graphs, the models, and the basic statistics (fraction of cells in G1, S, and G2). You can also copy the table of statistics to the clipboard by clicking on the button right above the table--and then paste into any spreadsheet or word processor for further analysis.
You may also wish to view the page on hints for performing Cell Cycle analyses.
Download a Cell Cycle Workspace with Demo Data to try out this platform.
