Overview of Figure Generation using the Working Illustration

Background

The Working Illustration is where figures (also known as Illustrations) are created and modified in Cytobank. The process of working with figures is accelerated by putting in effort ahead of time to annotate the experiment with Sample Tags. Learn about Sample Tags in the overview article here, or see a real example of applying Sample Tags in this article.

Sample Tags become variables within the Working Illustration. These variables can be toggled on or off and rearranged dynamically to build and modify a figure. This allows one to work above the level of FCS file names, and instead use the scientific variables that were present in the experiment, to build a figure.

Gating and compensation (if necessary) should also be done before creating a figure.

 

The Process - Outline

The process of creating a figure in Cytobank with the Working Illustration can be broken down into multiple parts. Clicking on the link for each part will jump the page downward to the explanation for that part.

 

1) Choose which Figure Dimensions boxes, and which contents of these boxes, should be active according to which data are desired in the final figure.

2) Configure the plot controls panel on the left side of the Working Illustration as desired for plots, statistics, equations (e.g. fold change), and styling (e.g. color, labels, plot size).

3) Update the illustration and dynamically pivot and optimize the layout and settings.

 

If you want to follow along, find the public Experiment called PBMC (fluorescence) in your Inbox on whichever Cytobank you are registered on and fully clone it. Open your Working Illustration inside the newly cloned Experiment (this is the default page upon completion of cloning).

 

Step 1) Choose which Figure Dimensions boxes, and which Sample Tags within these boxes, should be active according to which data are desired in the final figure.

  • When making a figure, the desired Figure Dimensions can be activated and deactivated by clicking on the buttons that indicate the name of the variable class (e.g. Conditions, Time Points, etc.).
  • The way in which the final figure will be arranged is dependent on the horizontal arrangement of these boxes. This will be optimized later by dragging the boxes left and right (see later in article).

 

 

  • After the desired dimensions are activated, the contents of the dimensions themselves need to be activated. When doing this, move from the left box to the right, and at each box, consider which variables should be present in the final figure.
  • To open a Figure Dimensions box and select elements to be part of the figure, simply click inside the box, or click the "setup" button in the upper right corner of the box. The contents of the box can be filtered to help find selections. The 'all' and 'none' buttons can be used to help select numerous selections, and these buttons can be used with a filtered selection.

 

(click to expand animation)

 

  • In the figure being created, functional readouts of phospho-STAT3 and phospho-p38 are desired on four gated populations across four stimulation conditions. This will create a figure with 16 principle data points (4 * 4 * 2).

 

Step 2) Configure the plot controls panel on the left side of the Working Illustration as desired for plots, statistics, equations (e.g. fold change), and styling (e.g. color, labels, plot size).

  • On the left side of the Working Illustration is the plot control panel. Starting from the top and working downwards, each aspect of how the figure will appear and report statistics can be chosen.
  • In the example below an illustration is being configured with:
  1. Plot type of density dot.
  2. Y channel of FSC across all plots.
  3. X channel selection of "Panel/Channel" Values, which creates a variable X axis among plots according to the Channels figure dimension box.
  4. A statistic of median for each plot type (calculated on X axis)
  5. A comparison equation of transformed ratio, which is a fold change in context of however the data are scaled (arcsinh in this case).

 

(the plot controls pane is home to all settings for the visual and statistical readout in the Working Illustration)

 

Step 3) Update the Illustration and dynamically pivot and optimize the layout and settings.

  • After any change is made in the Working Illustration, a green update button will appear in the bar along the top of the screen. For any changes made to the illustration to take effect, the update button must be clicked.

 (the Update Illustration button will appear when changes have been made to the illustration settings. Click the button to update the illustration according to the new settings)

 

  • Once the button is clicked, the illustration will update to show the effect of the latest configuration changes. In the case of the example shown, the layout is not ideal for interpreting the signalling data.

 

 

  • In order to improve the illustration layout, the Figure Dimensions boxes can be dragged and dropped to a different configuration. Below, the Channels dimension is moved to the right, which no longer emphasizes close comparisons between the members of that dimension within the Illustration. Instead, stimulation conditions will now be compared closely to gated populations, and two separate tables of this comparison will be displayed for each channel.

 

 (Illustration of rearranging Figure Dimensions to update the layout of the figure. Remember to click the button to update the illustration)

 

  • The figure will update immediately and the newly arranged data can be viewed. The figure below is much easier to interpret!

 

 (updated figure after pivoting using the Figure Dimensions. The layout of the figure is easier to interpret)

 



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