Data compensation is an important step in cytometry data analysis. Experiment-wide compensation is integrated into the Cytobank platform to help users manage compensation settings consistently across all analyses. In this article, you will learn how to modify a File-Internal Compensation matrix in the Cytobank platform, and the impact of modifying compensation throughout your analysis.
How to modify a File-Internal Compensation matrix?
Many users choose to calculate a compensation matrix directly on the flow cytometer prior to collecting samples. This matrix is written into the exported FCS files (File-Internal Compensation). However, sometimes it becomes apparent during analysis this computed matrix needs adjustment. To modify File-Internal Compensation, you first need to extract the matrix.
How to access the Compensation Editor
When you navigate to an existing Cytobank experiment, access the Compensation Editor by clicking the Compensation tab on the experiment level navigation bar. If the current setting is Uncompensated, the Compensation button will not appear in the navigation bar. In this case, please click Actions -> Edit compensation to access the Compensation Editor.
(Navigate to the compensation page within an Experiment)
Click the Import button on the page specific navigation bar.
(choose to import a comp from the compensation page)
Under the File-Internal Compensation tab, select a representative FCS file that contains the File-Internal Compensation matrix you wish to use and click Import Compensation. A new compensation matrix will appear on the page, named based on the file from which it was imported.
(Selecting to import a File-Internal matrix from a file)
From the list of compensation matrices, select the one that you extracted from the FCS file. To make edits, click inside any cell in the compensation matrix with a number and edit that numerical value. Changes will be saved automatically and reflected in the plot to the left. Alternatively, you can use the sliders to adjust the matrix values. This will automatically update the value in the matrix table.
(adjusting a compensation matrix manually)
In order to use this compensation matrix when gating and creating illustrations, ensure that it is selected as a compensation matrix under the Experiment compensation menu. Please read the next section before making any changes to experiment-wide compensation settings.
Impacts on Cytobank tools after changing the experiment-wide compensation settings
Many Cytobank tools are closely linked with an experiment-wide compensation setting that users choose in the compensation settings page. It is highly recommended that users select an appropriate compensation setting during the initial guided experiment setup process and do not change compensation during any downstream analyses, such as manual gating, creating illustrations, running advanced analyses, or exporting statistics. If the experiment-wide compensation is changed, the latest compensation settings will be applied when using all tools, except existing saved illustrations and gate definitions.
Please refer to the next section for more information about the impacts of a compensation change on the Illustration and Gating Editor.
After changing the selected experiment-wide compensation, all previously saved illustrations will still keep the previous compensation, and users will see a red warning in the compensation section of the More plot settings page from Plots>More plot settings in the Illustration Editor.
(A red warning will appear on the More plot settings page if illustration compensation is different from experiment-wide compensation.)
By clicking the red warning, users can decide whether to apply the updated experiment-wide compensation settings to the existing illustrations created with the original setting. It is recommended that users update the illustrations with the latest compensation setting. By default, Cytobank will not update illustration compensation to match it with the modified experiment-wide compensation.
The Gating Editor will automatically hide existing gates if users change the experiment-wide compensation setting. When clicking any hidden gates, users will see a red warning on the gating editor page.
(A red warning will appear in the gating active compensation setting section after users click any hidden gates.)
To unhide the hidden gates or remove the red warning, users need to go to the compensation editor page and restore the experiment-wide compensation settings on which the gate was drawn.
All other tools
All the other Cytobank tools automatically use the latest compensation settings after users make a change in the experiment-wide compensation setting page. Please see How the experiment-wide compensation work in Cytobank
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