FlowJo Software Lecture Outline:

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1 FlowJo Software Lecture Outline: Workspace Basics: 3 major components 1) The Ribbons (toolbar) The availability of buttons here can be customized. *One of the best assets of FlowJo is the help feature* 2) Group Section The power of analysis in FlowJo is organizing and analyzing your data into groups. Grouping your data allows you to apply common gates or analysis parameters specifically to a given group of samples. When dragging in data files, FlowJo will make a separate group for each data folder FlowJo will add a Compensation group automaticly and will often place samples here based on the FCS filename. If you compensation sample files were not detected automatically, you can add them manually to the group before making your compensation matrix. 3) Sample Section Contains a list of sample files by filename in the currently selected group. Working with the Workspace and Organizing Data: Adding Keywords: Click on sample circle to get access to keywords and parameters embedded within the FCS file *Parameters in this list can be edited on the flow cytometer only. Keywords can be added to groups or samples to allow further organization of your data and these keywords are then attached to this FCS file within FlowJo only! Files within a group can be organized by the added keyword. Graph Display: Double clicking on the sample in the sample list will bring up the graph display for that file. Here you can change the axis or parameter of the data that is displayed. If you close the window, FlowJo will automatically remember what display format you used last.

2 The two main modes of data display types are: 1) Univariate Histogram 2) Bivariatea) Dot Plot b) Contour plot c) Pseudo color density plot d) Gray scale density plot ** It is also possible to display your data in a 3D graph format** FlowJo by default should choose to display the data in a biexponential transformed format, which will allow values near to or below zero to be correctly visualized and gated. Axis scaling can be changed (but doesn t need to be) by clicking on the T box in the graph window. Gating: From within the graph window, a variety of gates can be drawn to begin subsetting your data. Once drawn, this gate will appear below the sample in the sample window. This gate can be renamed if desired. There is a variety of different types of gates offering different properties. The gate chosen should fit the type of data you are intending to acquire. Once the gate is created, it can be dragged to either another sample or applied to the group. After applying the gate to the group, the gate changes to the color txt of the group. If the gate is adjusted, its color will return to black, this adjusted gate can then be re applied to the group. To drill down into the data that you gated, click on the population you created and a new graph will appear of only that gated data. Changing the position of the original gate will update the data shown in the new window in real time. Adding Statistics: To obtain additional information about any population, you can add add a statistic to the population. Under the Workspace tab, you can find the statistics button. For example, you can obtain the Median Fluorescence Intensity (MFI) of a population. This statistic can then be applied to the whole group of samples.

3 Creating Data Tables with the Table editor: The next major section of FlowJo is the Table Editor, where data statistics and keywords can be combined and your data exported for further analysis and visualization To get the chosen data into a table, you drag the population or statistic of interest into the table window. You can also add keywords associated with each file/data point to be included in the workspace. To do so, click on Edit >Keywords *Although the data looks like rows, each row will form a column in the new worksheet. Rather than dragging in a statistic, you can also add a column to the table by clicking Add Column *Mathematical formulas can also be added to the table at this point The order of the columns can be sorted by picking up and dropping the column of choice. To get your data into its final table format is called Batching There are several different options for how to output your table data: 1) Batch to the clipboard for copy paste 2) To a file to open in Excel (usually a CSV format) 3) To the current Layout. 4) To the printer 5) To a figure (within Experiment Annotation) Tables are saved along with you workspace and you can make as many different tables as you need. Making data reports with the Layout Editor: The final major section of FlowJo is the Layout Editor, where you are able to save data visualizations for future reference or to generate data reports for exporting. Data that is displayed in the layout is in real time, unless you lock the content of the layout. Meaning that any changes you make to gate in the workspace will change the data that is displayed in the layout graphs automatically. Layouts can be named individually similar to the table editor. To get your graph into the layout editor, drag and drop your population of interest into the layout editor window. **Note: The graph that is copied over will be whatever type of visualization and axis labels you last used for that data. This can be changed by double clicking the graph within the layout editor as well.

4 It is also possible to changing additional format options for the graph by double clicking the graph in the layout editor. Keywords from your workspace can be displayed or added to your graph as well in different places As a text box, within the legend or in the sample information. Statistics can also be added to these same places. You can also return to your Table editor and batch your table to your current layout to include with your data. After arranging the graph and labels, you can now batch this layout for every sample in the selected group to make a report for all of your samples. Usually the best method is first batch to a new layout so that you have a copy of this layout saved within FlowJo. Then you can batch to either a pdf or powerpoint file. NOTE: Batching to a new layout is the only way to stay live or allow your data to be updated in real time. One specific type of graph that can only be visualized in the layout editor is the overlay graph. To create an overlay, drag the population of interest into the graph you want to overlay. The populations will be overlaid in the same graph. You can change the ordering or coloring of the populations. Overlays can also be created for histogram line plots. Within the layout, you can also easily generate a gating history to show all of the gates used for a given graph by second mouse clicking on the graph to pull up the options and choosing Ancestry Also within the Layout Editor are other drawing options to help arrange and align graphs and make more formal graphics. Saving the Workspace analysis: There are 3 main ways you can save you workspace: 1) Saving as a Workspace file (WSP) that does NOT contain the FCS files (but retains all of the analysis, gates, graphs). If the associated FCS files are within the same directory they will load into the workspace.

5 2) Saving as an Archive file (ACS) that contains both the FCS files and the workspace in a zipped archive file (A good option if you are planning on sending your data analysis to another colleague). 3) Saving the workspace as a Template file (WSPT) that only contains your gating, group organization and batched exports but does not contain any data. Saving a template for your analysis is useful if you are doing repeated analysis or repeating experiments and want to keep gating or gates the same.