Lesson 2: Flow Cytometry Analysis

While the software package utilized within the flow core is CellQuest Pro, the software is dongle-protected and quite expensive. As such, you will practice data analysis with a similar program known as Weasel.

Weasel is a flow cytometry data analysis program available for download from the Walter and Eliza Hall Institute of Medical Research. As it is java-based, it will run on most modern platforms. Please visit the Weasel download page and download the appropriate version for your computer. Please be sure to follow the installation instructions carefully. Java and Quicktime may also be required if not already present on your system. (Note: If you are running on an HITS-administered workstation, you may need to contact the help desk in order to have the software installed).

You may, of course, use any other cytometry analysis program which is already installed on your system. The University offers substantial discounts on many cytometry software packages. Be sure to contact M-Stores Customer Service prior to any purchase.

Finally, download the data files to be used for this exercise. To prevent corruption on download, the files have been archived in zip format. If you need the files in another format please email me.

Platform Tips

  • Windows: Depending upon your version of windows, weasel should be started by the Weasel.bat (win9x/NT) or the Weasel.jar (XP/2000) file.
  • Mac OSX: Weasel utilizes a two mouse-button scheme. When the instructions refer to a “right click”, simply press and hold the control or option keys while clicking to emulate a right click.
  • Linux/BSD: Functions identically to Windows version. Use the Weasel.jar file to start the application

Opening Data File
Open the weasel application. If you have purchased a license for the program, enter your registration code, otherwise click OK.

BRCF Flow Cytometry Training: Opening a data file for analysis

Note the pull-down menus within the program. Take the time to familiarize yourself with the various options under each menu. Weasel has a very rich documentation under the Help menu. If you are unsure or are curious about a feature, this is the first place you should look. Under the Edit menu is the Preferences option where you can customize the look and feel of the application.

From the Display Menu choose New Dot Plot. The file browser will appear. Locate and open the Norm.001 data file which you downloaded previously. The Dot Plot Format window will open. Choose the following options:

  • Parameters: FSC-H vs. SSC-H
  • Dimensions: Width 150 Height 150

Click OK, and the software draws a bivariate dot plot. The plot should be familiar to you from the previous lesson. Right-click in the center of the plot and choose Create a Region. Use the mouse to draw a region around the lymphocyte population (draw a polygon by clicking the vertices; click the starting point to close the region). In the Create Region dialog box, name the region “lymphs“. At this point, your plot should resemble this:

BRCF Flow Cytometry Training: Scatter plot from data file analysis

Go back to the Display menu, and again choose New Dot Plot. Choose Norm.001 and set the Parameters to Fitc vs PE. Click OK and view the new dot plot. Next, right-click in the center of the new plot and choose Change Format. In the Dot Plot Format window, choose Gate Create/Edit. A new dialog box opens (Create or Delete a Gate). Select lymphs under regions and click OK to close both dialog boxes. How is your plot different?

By creating a gate using the lymphs region, the new dot plot is only showing those cells which fall within the lymphs gate. Right-click on the plot again, and return to the Change Format window. Click the Gate Create/Edit button again, but this time choose the Not option as well as lymphs. Click OK to close the dialog boxes and look at your plot once more. How is it different? Right-click in the plot and choose the Gate option. Notice that you may now switch between no gates (None), the lymphs gate, and the NOTlymphs gate. This is a very useful technique when searching for various discreet subpopulations. Reactivate the lymphs gate.

Right-click within the Fitc vs PE plot and choose Set Stats Markers. The Set Quadrants dialog appears as well as a set of quadrant markers on the dot plot. Click on the quadrant markers and adjust them so that the lower left quadrant encompasses the first decade on the x and y axis. Observe the quadrant percentages and ensure that there is less than 1% total events in the remaining three quadrants. Click OK. Right-click in the plot and choose Set Stats List. In the resulting dialog box, choose the following options for the Quad Stats:

  • # Cells
  • % Totoal
  • % Gated
  • Mean X
  • Mean Y

Click Show Statistics. A statistics window appears attached to the dot plot. Right-click in the stats window and choose Detach Stats. Drag the stats window to a clear part of the screen and expand it so that it can be read.

The Quad Stat is the basic statistical measure within flow cytometry. By setting the stats on a negatively stained sample, we define the scale encompassing cells as negative and anything in the other quadrants as positive. Right-Click in the Fitc vs PE plot and choose Next File. Notice how the axis labels change (CD3 Fitc vs CD19 PE). Notice also that there are distinct CD3 and CD19 positive cells, but there do not appear to be any cells that are positive for both markers. What does that tell you about CD19 positive cells? Change the Gate to NOTlymphs. How does the dot plot change?

Wrapping Up
In order to asses your understanding of the material thus far, please email the answers to the following questions to the address below. After receiving this I will provide you with access to the next module.

  • Complete a Fitc vs PE sample for all twelve data files using both the lymphs gate and a new gate drawn drawn around the monocytes. Tabulate and Record the quadrant percentages (use UL,UR,LL, LR for quadrant designations) for each sample and each gate. Record your observations regarding the CD3, 4, 8 and 19 markers.
  • Return to the Display menu and experiment with the Histogram, 3D Dot Plot, and Contour Plot options. What benefits do you see to each of these options? When might they be useful?
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