Before you create a histogram overlay, you must have already: uploaded experiment files (or cloned those of another experiment); annotated your staining panels (channels); drawn your gates (created populations); and annotated any conditions, individuals, or other parameters that you want to use in your plots.
On the Illustration page, under "Plot Controls", select "Plot Type" "Histogram", check the Overlay option, and set the X axis to "Use Panel/Channel Values." and click "Update Illustration."
Make sure to select the appropriate channels for your histogram overlay in the "Channels" dimension. Click "Update Illustration." Adjust the other dimensions as needed and click on "Update Illustration."
Because this is a histogram overlay, the plot statistics area is active on the side bar under the illustration tab. In the plot statistics area, you can select to color your overlay using a particular statistic or normalize against an "Unstimulated" or control sample. Select "Fold" for equation and "First Row" (or whatever is appropriate) for control and click "Update Illustration."
A histogram overlay should appear in the illustration tab. The corresponding table of values will also be present under the histogram overlay and legend. This table can be exported as tab-delimited data by hovering over the "Export" section under the "Actions" dropdown menu on the experiment level navigation bar and clicking "Export Table of Statistics".
Once you are satisfied with the histogram overlay, you can save the illustration to get a persistent URL to share with others. Export the histogram overlay by generating a PDF of your illustration. Alternatively, you can open the heatmap in a print view and copy and paste the image into a program of your choice.
When any histogram plot type is selected, tools will appear in the "Plot Controls". For non-overlaid histograms, the aspect ratio, fill color, and outline color can be fine-tuned. Options to apply a highlight to the histogram outline, as well as to smooth the outline can be toggled on or off.
Overlaid histograms have the same styling options as non-overlaid and also gain a few unique parameters. For each histogram in an overlay, the precise vertical spacing can be defined and the presence or absence of a baseline can be toggled. Additionally, a "Statistic" option is added as a way to fill each histogram in an overlay. The fill color for this option is based on the statistic value calculation defined in the "fill" dropdown menu.