How can I create an unmixing matrix of autofluorescence, and import autofluorescence into EasyPanel?

If working with Cytek data, you may begin with step 1. If working with data from other instruments, ensure that you have already created the necessary ExtraKeywords file that defines a spectral parameter. Learn more about that process here.

Note: The EasyPanel Integration tool is available in FCS Express version 7.26 and later.

1. Select Tools tab, Compensation and Unmixing

2.  Click the down arrow in the upper-left corner, and select Unmixing.

3.  Under Step 1- Select single-stained controls, click the yellow file folder icon. Select a Raw unstained file that exhibits the autofluorescence of interest. Click Open file.

 

4.  Click the Sample Type dropdown, set the Unstained data file to Autofluorescence control.

5.  Click the Yellow file folder icon under Step 1 - Select single-stained controls. Select another data file that contains the same parameters as the first selected data file.
In this example, the same Unstained data file was selected again.

6. Click Open file.

 

7.  Under Sample Type column, set the second row to Autofluorescence control.

 

8. If not already done, enter a unique Label for each control. In this example, we have labeled to differentiate autofluorescence from monocytes and lymphocytes.

Optional: You may continue adding more data files to Step 1 section, if desired.

9.  Under Step 2 - Select Scatter Gate Parameters, select the preferred parameters to display on the X and Y axes when gating around the populations of interest. FSC-A vs SSC-A are typically recommended.
Under Step 3- Select Spectral Parameter, select the merged spectral parameter.

 

10. Automatically, FCS Express will create one layout page per data file. The pages are named according to the labels in Step 1 of the Unmixing Wizard.

• On the page for the first data file (e.g. "Lymphocytes" shown below), click on the "Scatter Gate", click the Gating tab and click Data Specific Gate.
• You may move and resize the "Scatter Gate" to the desired position.
• The goal is to select the population for which you want to quantify autofluorescence.
• Data Specific Gating allows the user to set a unique gate position for each data file.
• You may also adjust the Marker on the histogram to surround the population of interest.

 

11. Repeat step 10, but for each other data file. You can see below, that now the Monocytes page is displayed, and the Scatter Gate is again set as a Data Specific Gate for the Monocytes control.
The "Scatter Gate" has a unique position around the monocytes population.

 

 

12. In the Compensation and Unmixing window, click on the Unmixing Matrix tab. You may view the calculated unmixing matrix here.
One column of values per data file is calculated. To import the autofluorescence signatures to EasyPanel, click the green EasyPanel logo (inside the red box below).

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13. After clicking the EasyPanel button, click on the Autofluorescence tab. You may click on any of the autofluorescence controls to view its spectral signature. In the screenshots below, you can see the spectral signatures for the Lymphocyte and Monocyte populations in this example.

 

14.  If you have an EasyPanel account and you wish to plug the Autofluorescence spectral signature into the EasyPanel panel builder, click Design Panel Accounting for Autofluorescence (shown in the screenshot above).

15. After selecting the Instrument, and other desired markers to include in the panel, an optimized panel is automatically computed which accounts for the autofluorescence that you imported from FCS Express. You can see an example of this below. This panel was designed to include a CD4 antibody, CD8 antibody and a Viability dye.

 

16. In FCS Express, a page showing the Visualized Mixing Matrix gets automatically added to the layout. This is another place you may view the normalized spectra of your data files.