FlowJo Standard Operation Protocol Basic Operation

Transcription

1 Launching FlowJo analysis software: FlowJo Standard Operation Protocol Basic Operation Log in Mac OS system with account of FlowJo user Download your FCS files from FCF storage server to the folder of users data for FlowJo analysis on Desktop. Please create a folder under your name if needed. Please go to: or follow laminated instruction on the table to download your data. Notice: No data is allowed to save in other directory on FlowJo computer. You are strongly suggested to read PDF files in the folder of FlowJo Tutorial and watch videos in the folder of FlowJo Tutorial Video on desktop before you start to use FlowJo to analyze data. Introduction There are 6 Steps to analyze data by FlowJo: Load data into the Workspace. Analyze the control tube data in detail (gate subsets and add statistics). Copy gates and statistics to All Samples. Verify gates on All Samples. Generate a graphical report including All Samples. Generate a table of statistics from All Samples. Step. Load data into the Workspace. Double click FlowJo alias 7.6 on desktop to start FlowJo program. When you open FlowJo, you will see the Workspace window pictured below.

2 Create New group in the Groups Panel by clicking Add Groups. Add group name in Name then click Create Group. Drag and drop FCS files or experiment folders (no need to create groups) to the Workspace window.

3 Double-click Tube 00 to open a graph window. Click the options triangle () to edit graph types. Click the drop-down box () to choose a new graph type. Click the Smooth box (only while displaying a Histogram or Pseudo- color). Use Color boxes (3) to change the fore- ground and background colors. Change the graph s axis parameters by clicking on the axis label () to pop up a menu of options. Select log or linear scale by clicking on beside axis label (6). Graph Type 3 Color box 6 3

4 Step. Analyze a sample in detail. Gating Draw a polygon gate encompassing the interested population. Name this subpopulation if needed. Double-click on the FSC-SSC subset gate in the Graph window to open a new graph displaying only the subset of cells that fall within the FSC-SSC subset gate. Draw gates for FSC-H/FSC-W subset and SSC-H/SSC-W subset if needed. The parameters that are displayed in the graph are easily changed by clicking on the X or Y axis labels and selecting another parameter from the pulldown menu. Double-click on the singlet subset gate (such as SSC-H/SSC-W subset gate). Click on the X-axis label and select fluorescent channels (such as, FITC-A). Click on Y-axis label and select Histogram from the menu if needed. Choose the Range Gate tool and draw a gate to set the range of fluorescence values that will define FITC positive cells. FlowJo shows the new subsets in the Workspace as a Gating Tree. The gating tree shows Grandparent, Parent, Child and Grandchild relationships between cell populations. A petri dish icon appears to indicate a subpopulation of cells.

5 Adding Statistics There are many statistics that can be computed and displayed by FlowJo, including fluorescence intensity (such as mean and median value), CV or frequency and more. Select the interested subpopulation in the Workspace () (row is shaded). Click on the Add Statistic () function button. This brings up the Add Statistic dialog box. Select the statistics (3) in the dialog box, such as Median or Frequency. Click the Parameter () drop-down menu and choose which channel to compute. Click the Add button (). In the Workspace, new rows of statistics are added under the subpopulation. Add statistic 3 Step 3. Copy gates and statistics to All Samples. Analyses can be applied to every sample simultaneously by dragging the gating tree onto a group name in the upper panel of the Workspace window. Select all the gates and statistics (Shift-click) and drag them onto the group in the upper panel of the Workspace. Gates and Statistics are applied to All Samples

6 Step. Verify gates on all samples. Checking Gates FlowJo does NOT check the gates on each sample for you! To check your gates, you can click on the forward navigation arrow at the top of the Graph window to check the gate on each sample. You can check multiple graphs of any sample simultaneously. In order to advance the gates in both graphs at once, shift-click the arrow. All open graphs of tube cycle to graphs of the next sample. Shift-click Altering Gates Gates could be altered on each individual sample but its easier to adjust one gate and apply the new region to all the samples by drag-and-drop. Drag-and-drop to apply it to all samples Altered gate is turned into black 6

7 Step. Generate a graphical report. Layout Editor Open the Layout Editor via the Layout button at the top of the Workspace window. In the Workspace Window, drag the interested subpopulation from Tube 00 to the Layout Editor. A corresponding dot plot/histogram will appear. (Double-click on the graph in the Layout Editor to edit parameters displayed in the graph.) In the Workspace Window, select the statistics under Tube 00 and drag them to the right panel in the Layout Editor. 7

8 Adjust size and layout of all graphs of one sample on layout editor. Make a batch report: Click the drop down box and select Sample. Then click on the Batch button in the upper right corner of the Layout Editor. If you would like to save batch analysis layout in PNG file, click save tab. File name Directory Batch analysis layout could be generated into PPT or PDF files. Chose Powerpoint or PDF for batch report, then click Create in the dialog window. A new file (PPT or PDF) is created. The batch report of PDF file is shown below. The layouts can be saved to disk or copied and pasted into many drawing programs as well. 3 File name Directory 6 8

9 Histogram Overlay Create a new layout by clicking the New Layout button (+ sign) in the Layout Editor. Drag the subpopulation of Tube 00 from the Workspace to the Layout Editor. Adjust if necessary to display a histogram. Control-click the SSC-H/SSC-W from Samples through and drag them all on top of the graph already in the Layout Editor. When you release the mouse button, the SSC-H/SSC-W subpopulation from every sample will be overlaid in one plot. When a large number of histograms are overlaid, Right-click on the graph and select the Staggered Offset option from Histogram. Your graph will look like the one below. 9

10 Step 6. Generate a table of statistics. Open the Table Editor by clicking the grid icon. In the Workspace Window, shift-click the interested subpopulation and statistics from Tube 00. Drag them to the right panel of the Table Editor as shown below. Add a name for your table in the text box at the top of the Table Editor Window if necessary. If you would like to add more statistics parameters into table, click the column info button to open the Column dialog. Add new statistics parameter then click OK. 3 Create the table by clicking the Batch button in the upper right of the Table Editor. 0

11 Chose FlowJo or File for batch report, then click OK in the dialog window. A new layout (FlowJo) or a new file (Excel) is created Save workspace Users are strongly suggested to save workspace when analysis is completed. The workspace file will save all gate settings for you future reference. File name Directory