Web-based analysis and publication of flow cytometry experiments

Abstract

Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org.

Figure 1.1.1

Searching the Experiment Inbox for the “Welcome to Cytobank – U937 dataset” experiment. In the upper right hand corner search box, type “welcome” to filter the experiments and show only those which match that text. https://www.cytobank.org/cytobank/experiments

Figure 1.1.2

A histogram overlay figure on Cytobank. This figure shows the response of the U937 cell line to various stimulation conditions (in rows) at different signaling node readouts (p-STAT6, p-STAT3, p-STAT1, and p-STAT5, in columns). Histogram overlays on Cytobank shade peaks according to a statistic defined by the researcher. In this histogram overlay of a signaling experiment, the peaks are shaded by the fold change of each stimulated sample from the unstimulated control (in the first row). Each column shows intracellular detection of a phosphorylated STAT protein (1). Also included is a table of the statistics and a color scale. https://www.cytobank.org/cytobank/experiments/61/illustrations/96#Illustration

Figure 1.2.1

Downloading files from Cytobank. To download files from Cytobank, click the “Download FCS Files” link and follow the instructions to select a folder on your personal computer and start the download. This download will retrieve entire files in FCS or text format. Download of gated subpopulations can be done from within illustrations. https://www.cytobank.org/cytobank/experiments/61/download_files

Figure 1.3.1

Creating a new experiment on Cytobank. Prior to upload, organize your files into groups by experiment. You give each experiment a name and can assign it to a project. You also describe a short purpose for the experiment. These details can be searched later, if you are trying to find an experiment. https://www.cytobank.org/cytobank/experiments/new

Figure 1.3.2

Successful file upload. Following file upload, you should see this message and links to either start an illustration or view the experiment summary page. Below the message you will see your files categorized and a plot of Forward and Side Scatter for all the files to make sure each file is readable.

Figure 2.1.1

This figure shows the configuration of the controls and the resulting contour plot illustration at the end of the Contour Plot protocol. (A) Configuration of the controls to make this illustration. All the dosages are selected and this dimension is shown in the columns. For other dimensions, just one entry is selected to keep the figure small. (B) Contour plots show an IL-2 dose response where p-STAT5 was measured in CD3+ T cells. The x-axis shows CD4 expression and the y-axis shows phosphorylation of STAT5. The doses are arranged in columns, starting with unstimulated cells, then a dose response from 100 ng/mL to 0.01 ng/mL. https://www.cytobank.org/cytobank/experiments/310/illustrations/583#Illustration

Figure 2.2.1

Setup of the figure dimensions for a histogram overlay comparing dosages. When the plot type is a histogram overlay, the second dimension is “Overlaid”, which means that it forms the overlaid peaks within the histogram overlay. Usually the overlaid dimension compares conditions, individuals, timepoints, or dosages.

Figure 2.2.2

A histogram overlay showing the dose response for CD3+ T cells from two healthy blood donors. Along with the histogram overlay is a table of data showing the fold change calculations. https://www.cytobank.org/cytobank/experiments/310/illustrations/584#Illustration

Figure 2.2.3

A table of fold change statistics exported from Cytobank. This table of statistics compares the median fold change of each peak of CD3+ T cells stimulated with a different dose of IL-2, relative to the unstimulated state.

Figure 2.3.1

Order of the dimension controls to create a heatmap of doses. For a heatmap, we moved the dosages into the columns and the individuals into the rows.

Figure 2.3.2

Placing the mouse over a square of a heatmap shows a view of the underlying data. Shown here is a heatmap view of the dose-response experiment. The user moves the mouse over one square for 0.07 ng/mL IL-2 in Donor 2, showing a 2D density dot plot view of the data used to generate the statistic in that square. This type of view through keeps users connected to the data, while also providing a high-level summary of the results. https://www.cytobank.org/cytobank/experiments/310/illustrations/585#Illustration

Figure 3.1.1

Web based gating. This figure shows drawing a polygon gate around intact cells using a plot of side scatter vs. forward scatter. https://www.cytobank.org/cytobank/experiments/310/draw_gates

Figure 3.1.2

An automatically generated layout checking whether the Intact Cells gate ‘fits’ for all of the files in the Phospho-Flow Cytokine Titration experiment. In this case, one global Intact Cells gate is a good fit for all the files and identifies ~90% of the events as intact cells in all the files. https://www.cytobank.org/cytobank/gates/check_gates?experimentID=310&gateID=1&populationID=0&plotType=14

Figure 3.1.3

Figure 3.2.1

Clicking the “Individuals” button activates this dimension for annotation. The dimension is initially red, indicating that files need to be annotated for “which individual this file matches”.

Figure 3.2.2

Annotating information about ‘Individuals’. The tags “Donor 1” and “Donor 2” are entered as a comma-delimited list into the Individuals annotation page. This creates two individual entries (Donor 1 and Donor 2) and looks for files with matching text in the filename or sample ID.