Getting Started in FCS Express for Previous FlowJo Users

Transcription

1 Getting Started in FCS Express for Previous FlowJo Users The instructions below emphasize similarities and differences between FCS Express 5 and FlowJo v10 and are meant to serve as a translation guide for those familiar with FlowJo and working with FCS Express for the first time. Each section displays how an action is handled in FlowJo and how that similar action is completed in FCS Express. At the most basic level, most actions in FCS Express are designed to act and feel just like creating a PowerPoint presentation. Once your presentation (called a layout in FCS Express) is created, you may change data files on plots to quickly review and batch export multiple files to multiple reporting formats with a few clicks. The layout may be saved for later use with new data files from a similar experiment or modified for new experiments. The De Novo Software team is committed to your success. Along with the instructions below there are a wealth of learning resources available to you on the De Novo Software website Here you may access: Tutorials Searchable Manual Recorded Webinars and short instructional videos Many of our recorded webinars are presented in multiple languages on the De Novo Software YouTube Channel. If these resources are not enough please do not hesitate to contact our expert staff to ask questions or schedule a free online training session at: support@denovosoftware.com *FlowJo is a registered trademark of FlowJo, LLC *PowerPoint is a registered trademark of Microsoft Corp. Page 1 of 13

2 Section 1: Loading your flow cytometry data into the software FlowJo (FJ): Samples are added to a workspace by dragging and dropping files from a system folder to the workspace or by using the Add Samples button in the Navigate ribbon (Figure 1). Figure 1: Loading flow cytometry data files into FlowJo. FCS Express (FCSE): Files are added to the Data List (accessed through the Data tab Organize Data Sets Data List) by dragging and dropping files from a system folder on your computer into the Data List. Files can also be added by clicking on the green + sign in the Data List (Figure 2) and navigating to the file location. Figure 2: Loading flow cytometry data files into FCS Express. Page 2 of 13

3 Section 2: Opening a sample data file FJ: Data files in the workspace are opened by double clicking on the file in the workspace. Double clicking will open up the file in a separate window called the Graph Window (Figure 3). Figure 3: Displaying a flow cytometry data file in FlowJo as a plot. FCSE: A data file is displayed by dragging and dropping the file from the data list to the layout or choosing to insert a plot type from the Insert tab. When dragging and dropping, the Select plot types window will open and allow you to insert one or many plots types at the same time. To insert multiple plot types, check the boxes for the plot types to display and click OK. The file will now open in the chosen plot types (Figure 4). When choosing to insert a plot from the Insert tab (red box), click on the plot type from the Insert tab (green box). Then click on the layout or draw a box as you would for inserting an object in PowerPoint. The plot type you selected will now appear on the layout with the data you selected displayed in the plot (Figure 4). Figure 4: Displaying a flow cytometry data file as a plot in FCS Express. Dragging and dropping a file allows you to select multiple plot types while using the insert tab creates one plot at a time. Page 3 of 13

4 Section 3: Changing between different plot types FJ: Plot types are selected and changed through the Options tab in graph window using the Type drop down menu (Figure 5). Figure 5: Changing a plot type in FlowJo. FCSE: Select one or multiple plots, as you would with PowerPoint objects, and choose the Format Tab Change Change Plot Type drop down. Choose any plot type from the drop down list to change the plot type on the selected plots (Figure 6). More commonly in FCS Express you would directly insert the plot type of choice directly on the page from the Insert tab. Figure 6: Changing a plot type in FCS Express. One or multiple plots may be selected, just as you would select multiple objects in PowerPoint, and changed at the same time. Page 4 of 13

5 Section 4: Changing axes parameters. FJ: The X and Y axis parameters may be changed in the graph window by clicking on the X or Y axis and selecting the new parameter (Figure7). Figure 7: Changing axes parameters in FlowJo. FCSE: The X and Y axis parameters may be changed on the plot by clicking and holding on the X or Y axis. Choose the new parameter and release the mouse button to display it on the plot (Figure 8). Figure 8: Changing axes parameters in FCS Express. Page 5 of 13

6 Section 5: Gating on populations of interest FJ: Gates are created on a population of interest by choosing a gate type from gating tools in the graph window and drawing the gate in the graph window (Figure 9). Figure 9: Gating on populations of interest in FlowJo. FCSE: Gates are created on a population of interest by clicking on a gate type from Gating Tab Create Gates. Select the gate type from the ribbon and draw a gate on the plot (Figure 10). Figure 10: Gating on populations of interest in FCS Express. Page 6 of 13

7 Section 6: Applying gates FJ: Gated populations are further analyzed by double clicking on the gated population in graph window or on the population node in the workspace. This action opens up the gated population in a new graph window, where you can perform further analysis or gating (Figure 11). Figure 11: Applying gates to other plots in FlowJo. FCSE: A gate may be applied to any plot in four ways. Dragging a gate from one plot to another plot (Figure 12 green arrow), dragging a gate from the Gate View (found in the Insert Tab General Gate View) to an additional plot (Figure 12 pink arrow), and opening a new plot created (and gated automatically) by dragging a gate out of a plot and dropping it on the layout (Figure 12 blue arrow). Gates may also be changed by selecting a plot and choosing a gate, or no gate, from the Current Gate drop down (Figure 12 red box and arrow). Figure 12: Applying gates to other plots in FCS Express. 1. New plot created and gated by dragging a gate out of a plot and dropping it on the layout (blue arrow). 2. Dragging a gate from one plot to another plot (Green arrow). 3. Dragging a gate from the Gate View to an additional plot (Pink arrow). 4. Selecting a plot and changing the gate in the Current Gate drop down (Red box and arrow). Page 7 of 13

8 Section 7: Hierarchical Gating FJ: Hierarchical gating is presented in the workspace. When a new gate is created, the workspace shows the gate name below and indented from the sample on which it was drawn. In this way, you can follow the sequence for gating hierarchies by indentation. Indentations also indicate which gate is applied to a particular subpopulation. The hierarchy can be changed at any time by dragging and dropping the nodes throughout the hierarchy (Figure 13). Figure 13: Hierarchal gating within the workspace in FlowJo. FCSE: Gating hierarchies are displayed and changed using the Gate View (Insert Tab General Gate View) (red box). Indentations represent child gates and a subhierarchy may be collapsed or expanded by click on the +/- button next to the gate name (red box).the gating hierarchy may be changed by dragging and dropping gates throughout the hierarchy (Figure 14). Figure 14: Hierarchal gating in FCS Express is displayed an adjusted within the Gate View. Page 8 of 13

9 Section 8: Changing Axis Scaling FJ: The scaling of a plot s axes can be changed between log, linear, biexponential, archsinh, hyperlog, logical or miltenyi using the T button on the X and Y axes to reveal the scaling drop down. The biexponential, archsinh, hyperlog, logical or miltenyi options are available under Customize Axis (outlined in green) (Figure 15). Figure 15: Changing axes scaling in FlowJo. FCSE: The scaling of a plot s axes can be changed to log, linear, biexponential, hyperlog, lin/log, log with negative or Arcsinh by selecting the plot and accessing the Format Tab Plot Options Axes in the ribbon. Alternatively, right click on the plot and choose Format Axes category. Choose the Axis you wish to change (blue box) and the desired scaling from the Scale drop down menu (Figure 16). Figure 16: Changing axes scaling in FCS Express through the formatting Axes window. Choose the Y or X axis (Blue box) and choose the scaling from the Scale category. Page 9 of 13

10 Section 9: Creating overlays on histograms or dot plots FJ: To create an overlay, drag a file or population from the workspace into the layout editor. Then drag a second file or population on top of the graph in the workspace to create an overlay. You can also create an overlay by dragging-and-dropping from one plot in the layout editor onto another plot. These actions may be applied to histograms or dot plots (Figure 17). Figure 17: Creating overlays in FlowJo. FCSE: Overlays may be created in three ways: 1. Right-click on the plot and select Add overlay from the pop-up menu, 2. Drag-and-drop file(s) from the Data List onto the plot you wish to add an overlay, 3. Drag-and-drop a plot from the layout onto the plot you wish to overlay. Overlays may be created for either histograms or dot plots (Figure 18). Figure 18: Creating overlays in FCS Express. Data files from the data list were dragged and dropped onto a plot in the layout to create histogram and dot plot overlays, one of three methods to easily create overlays. Page 10 of 13

11 Section 10: Boolean gating FJ: Boolean gates are created by clicking on the Boolean button (Figure 19 red box 1). After creating gates in the workspace (Figure 19 red box 2), highlight the gates for use in the Boolean function and click on Boolean button (Figure 19 red box 1). Subsequently select the Boolean operator for the desired gate. In the example below Create Combination Gates (Figure 19 red box 3) was selected to create all possible combination gates (Figure 19 green box). Figure 19: Creating Boolean gates in FlowJo. FCSE: Boolean gates are created within the Gate View (Insert tab General Gate View). In the Gate View, highlight gates by through multiple selection (Figure 20 pink box) for which you want to create combination gates using Boolean functions. Rightclick on the highlighted gates and select Add Combination Gate from the pop-up menu. The Create combo gate dialog will open to create combinatorial (Boolean) gates (Figure 20 blue box). Type the gate name and Boolean functions such as and, not, or in order to create combinational gates. In addition, if Add Combination Gate Matrix option is chosen from the pop-up menu, then all possible combinational gates will be created (Figure20 green box). Figure 20: Creating Boolean gating in FCS Express. Select gates in the gates from the gate view and right click to access combination (Boolean) gate options. Page 11 of 13

12 Section 11: Batch process your analysis FJ: Multiple files are analyzed using the batch report iterative options. A batch report is accomplished by dragging and dropping plot(s) from one sample in the Workspace (Figure 21 red box) to the Layout Editor. The New Layout drop down in the Layout Editor window (Figure 21 blue box) is used to choose your report type. The Create Batch Report button (in green box) is clicked to finalize the process and will generate reports for multiple samples based on the first sample you placed in the Layout Editor (Figure 21). Figure 21: Batch analysis in FlowJo. FCSE: To batch analyze multiple data files first create an analysis for one sample in the layout (multiple samples or tubes may also be used). Open the Batch Actions window (Batch Batch Processing Batch Actions) (Figure 22 pink box). In the Batch Actions window, add batch process actions for the desired output/action (i.e, Print, PPT, XLS, PDF, DOC ) (Figure 22 green box). Once a list of batch actions is created, click the Run button (Batch Batch Processing Run) (blue box). Any actions you have included in the Batch Process Actions list (green box) will be completed for as many files as you have in your data list (Figure 22). Figure 22: Batch analysis in FCS Express. Batch actions allow multiple format exports for many files with one click. Page 12 of 13

13 Section 12: Exporting Statistics to Excel FJ: Statistics from multiple data files are collated using the Table Editor. Drag and drop statistics from the plot(s) of one sample to the Table Editor from the Workspace (Figure 23red box). Statistics are sent to the clipboard, printer, Current Layout, Figure and to Files by using any of the actions in the Table Editor window (Figure 23green boxes 1 and 2). In the To File option, you can choose the File format, in which you want to save your statistics (Figure 23green box 2). Choosing a file option will generate statistical reports for multiple samples base on your design in the format as previously selected (Figure 23). Figure 23: Batch reporting of statistics in FlowJo. FCSE: Statistics to be exported are selected using the Export to Excel Batch Action. Create an analysis for one sample in the Layout (multiple samples or tubes may also be used).the Export to Excel batch action should be selected to create the Excel format for which the statistics should be batched processed (cell mode or/and column mode) (Figure 24 blue box). With the Excel report action created, drag and drop the required statistics from the layout to the Excel action created in Batch Actions window (Figure 24 red arrows). Simply click Run (Batch Batch Processing Run) to export your statistics in the formats as selected (Figure24). Export to Excel may also be used in conjunction with any other batch action for complete one click reporting. Figure 24: Batch analysis of statistics in FCS Express. Page 13 of 13