Colored Overlay Dot Plots and Backgating


A useful tool in the analysis of cytometry data is the ability to overlay plots that would otherwise be viewed separately. The overlaid plots can be colored according to different gated populations or samples within an experiment.

blobid0.png (pSTAT3 expression is compared between different populations gated from PBMC stimulated with IL6 and IL10. Separate plots can be easily converted into a colored overlay for a different visualization) 

In the Cytobank platform, any dimension can be used to create a colored overlay figure. See how timepoints stack up or compare donors or doses. Overlay populations to backgate for immunophenotyping or to visualize small populations of cells among a greater overall background.  


(A rare signaling population is visualized in a larger context.) 


Pair colored overlay dot plots with viSNE to inform the quality of a manual gating strategy by comparing the location of colored events from gated populations to their location on top of a viSNE map.  Here we used viSNE as an example but the same workflow can be applied to other DR algorithms.


(An incomplete collection of manually gated populations are overlaid on the corresponding viSNE map of CD45+ PBMC (“Ungated” after viSNE). Blue events were not captured by manual gates, showing shortcomings in the gating strategy)


How to Make a Colored Overlay Dot Plot 

To create a colored dot overlay, navigate to the Illustration Editorselect a plot type of dot and turn on the Overlaid buttonAn overlaid figure will be automatically created with the default layout settings, which sets FCS Files in the Overlaid position and Channels in the Column position.  To overlay manually gated populations onto the dot plot, simply click on Layout in the Illustration Editor navigation bar, click on FCS Files, and select the box next to Populations to switch the manually gated populations to the Overlaid position. The ordering of variables in an overlay plot determines the stacking order and colors of those variables.   There are 3 options to set up the order: 


1) On the layout preview in the layout tab, deselect and select the new order (the first that you click will be in the back/bottom of the stack).  

2) Just click and drag one of the overlaid features on the overlaid legend on the left of the plot.  

3) Hover the mouse over the overlaid legend and click on the number of selected populations/files. 


If the gated populations are overlaid the hierarchy of the gates will be restored on the overlaid stack (ungated at the bottom, parent population, daughter population, grandchild population on top). If another dimension without a hierarchy is selected as the overlaid one, for instance, FCS files, then when you click on the number of selected files, the order you use when selecting the files will be the order of the overlaid stack. In this case, you can also click and drag the name of the files in that menu to choose the order that you prefer. The dot size and dot shape can be configured.


(The Illustration Editor configuration for colored dot plot overlays. As is the case with other dot plots within the Cytobank platform, the size and shape of each dot can be configured. Other relevant controls are shown, such as the X and Y axis settings)


 Customizing Colors 

In the Cytobank platform, you can customize plot colors by clicking on the color legend rectanglesYou can either pick a color from the color palette or enter your own RGB color code.



By default, dot coloring has an opacity of 100%. You can configure the opacity from 0 to 100 from the Plot Settings accessible from the navigation bar (shown on left) and by hovering over the plot (shown on right). 


(Plot Settings accessible from the navigation bar      Plot Settings accessible from hovering over the plot)


When the opacity is mid-range, you can see the other dots or lines behind the dots or lines in front, representing densityYou can save and load overlay coloring and styling preferences as part of Illustration Templates. 

Get in touch with Cytobank Support if you need more assistance. 

Video Introduction to Colored Overlay Dot Plots 

                           (video introduction to colored overlay dot plots)


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