Create a compensation matrix automatically from single stain controls (automatic compensation)

If you didn't take care of compensation at the cytometer, you might wish to generate your compensation matrix in the Cytobank platform from single stain controls. The Cytobank platform will automatically compute the compensation matrix using these controls via the Automatic Compensation tool. To access the Automatic Compensation tool, access the Compensation page from the Compensation tab on the experiment level navigation bar. 


Once you arrive on the Compensation page, click the Automatic button next to the Create new compensation. The Automatic Compensation page will pop up. To learn more about other ways to create new compensation, please see this article: Ways to create a new compensation matrix
Video tutorial on the automatic compensation

4 steps to create the automatic matrix 

1)You can select the channels that will be used to draw the cleanup gate. The cleanup gate is used to gate the events that will be used to calculate the compensation matrix. The default setting is FSC-A on X axis and SSC-A on Y axis. The Y channel you selected will also be the one used for the positive and negative gating plots. 

2)The next step is to select the compensation channels. Select the channels that are used for single staining to generate the matrix.  
3)Then select the files that contain the positive and negative populations for each channel. You may use separate files or the same files for each positive and negative pair. The Cytobank platform will auto-match the channels and files if the file name has the channel’s short name (i.e., the machine detector name). When ready, click to calculate the compensation matrix. 

4) You will be promoted to the calculated compensation matrix page. The newly created compensation will show up in the compensation table, where all compensation matrices are listed. The preview plot allows you to visualize the calculated matrix on the experiment samples. Within the preview plot you can change the FCS file that you’re previewing by clicking into the File/Sample Name box at the top of the plot.  You can change the channels associated with the X and Y axis by clicking into the appropriate box and changing to the channel that you want the plot to display. You may choose a gated population for your preview plots from the Population box. You can also change to a different compensation for comparison.  

Please note: if you adjust with the cleanup gates (see below), and refresh the compensation, any change to the preview plots settings will be lost and you must redo the settings with each cleanup gate change. It is recommended to modify the cleanup gate as needed before making changes to the preview plots. 


Pairwise plots with sliders to view and finetune the matrix 

The preview plot when displayed in the compensation channels on X and Y, also has slider bars to help you with adjusting compensation for your analysis.  Adjustments made to the compensation using the slider will be automatically calculated in the matrix table to the right of the preview plot. You may change the name of the compensation matrix by typing in the text box above the matrix table. 


(The slider bars on the preview plots allow you to finetune the compensation matrix value manually) 

Above the preview plot, you may click and open the Pairwise plot view. It opens the pairwise compensation plots page where you can view and adjust all your biaxial compensation plots at the same time. Each of the biaxial plots has its own sliders, just like with the preview plot. Adjustments made in the Pairwise plot view will be reflected in your matrix table. 


The sliders run between compensation values 0 to 100.  If you need to use negative compensation values or a value over 100, then you’ll need to make the adjustment from within the matrix table.  You can click on any value in the table to edit the compensation for that channel.  Hovering over a channel in the matrix table will reveal the reagent name used for that channel. 


Ability to manually adjust the cleanup and positive negative gates 

Down below are the Cleanup Plots where you can adjust the gates to define the events used for compensation. You must adjust the cleanup gates on a per-file basis. Unfortunately, we do not support global cleanup gates; please contact Cytobank Support if you’d like this feature to be added.   The positive and negative gates on the single stain controls are automatically generated with the ability to adjust the gates manually. After all the adjustments, you may click on the Refresh button to recalculate the matrix.  


(Manual adjustment of the cleanup gates and positive and negative gates) 

When you have finished, the newly calculated compensation matrix will appear under the list of compensations that you can select as experiment-wide compensation, and can select from the Illustration Editor, within the Gating Interface and Advanced Analysis.  


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