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Flow Cytometry at San Jose State University

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Paint-A-Gate™ Tutorial

Table of Contents:
1.0 Introduction
1.1 Conventions used in this document
2.0 Basic Principles of Flow Cytometry Data Analysis
2.1 Data Collection
2.2 Plotting data - histograms
2.3 Plotting data - dot plots
3.0 Using Paint-A-GateTM
3.1 Launching Paint-A-GateTM
3.2 Loading and Unloading FCS Files
3.3 Working with Plots
3.4 Creating a New Dot Plot
3.5 Painting a Population of Events
3.6 Gating a Population of Events
3.7 Zapping Painted Events
3.8 Displaying Data for Painted Events

1. Introduction

This tutorial will introduce the user to the basics of flow cytometry data analysis using the program Paint-A-Gate (BD Biosciences). It is assumed that the user is already familiar with the concepts of fluorescence and fluorescent labelling, and understands in a general way how a flow cytometer works. This material is covered in our introduction to flow cytometry and fluorescence microscopy section.

1.1 Conventions used in this document

Section 3 of this document contains both explanatory background material and instructions for the user to follow. To aid the user in visually distinguishing these, explanatory material has been enclosed in boxes such as the one that follows:

Boxes like this are used throughout the page to enclose explanatory or background material.

Menu commands or dialog box options selected by the user are printed in Chicago font. The pipe symbol, |, is used to set off sequential selections. Therefore, the instruction "Select File | New PAG Set" should be read as, "Select the File menu, and then choose New PAG Set from that menu."

2. Basic Principles of Flow Cytometry Data Analysis

2.1 Data collection

Each cell or particle recorded by the flow cytometer is referred to as an event. Six different types of data may be recorded for each event: side scatter (SSC), forward scatter (FSC), and up to four separate wavelengths of fluorescence. Paint-A-Gate reads data files that have been stored in a special proprietary format. It refers to these as FCS files.

2.2 Plotting data - histograms

The simplest type of plot used in the analysis of flow cytometry data is the histogram. A histogram displays a range of values for a parameter along the X-axis, and the number of events exhibiting each parameter value on the Y-axis.

Figure 1 shows a histogram of FSC values from a data set measured on a mixed population of Tetrahymena and fluorescein isothiocyanate (FITC)-labeled yeast. The histogram shows clearly the two types of cells present: the yeast, with small size/FSC and the Tetrahymena with larger size/FSC.

Figure 1. Histogram of FSC values in a dataset, with FITC-labeled yeast shown in green and Tetrahymena shown in red.

Although histograms are useful for showing the distribution of values of a single parameter in the population, the real power of flow cytometry comes from its ability to measure several parameters simultaneously for each event. To plot two parameters for a population of events, a dot plot is used.

2.3 Plotting data - dot plots

A dot plot is a plot in which values of one parameter are plotted against values of a second parameter. Each dot on the plot therefore represents a pair of values for a single event.

Figure 2 shows a dot plot of SSC plotted against green fluorescence (designated FL1-H). Again, two populations can be clearly distinguished: yeast, with low SSC and high fluorescence, and Tetrahymena, with high SSC and low fluorescence.

Figure 2. Dot plot of SSC vs. FL1-H for a dataset, showing two populations of cells: yeast (green) and Tetrahymena (red).

3. Using Paint-A-Gate

3.1 Launching Paint-A-Gate

To launch Paint-A-GateAcademic, click on the Paint-A-Gate program icon:

Note: If your monitor is not set to display 256 colors, a dialog box will appear asking if you want to reset it. Click on "Set monitors to 256 colors" to continue.

3.2 Loading and Unloading FCS Files

To load an FCS data file for processing:

1)     Select File | New PAG Set. This produces no visible change on the screen, but readies the program to begin analysis of new data.
2)     Select Process | Load FCS File. A file selection dialog box will appear on the screen. Locate and select the data file you want to analyze, and click Open.
3)     Six automatically generated default dot plots will appear.

To unload an FCS file and clear the program's memory to allow a new file to be loaded, select Process | Unload FCS File.

3.3 Working with Plots

When Paint-A-Gate loads an FCS file, it generates six default dot plots from the data. The user can close unneeded plots. Paint-A-GatePro also allows the user to move and resize plots on the screen.
To close a plot, click the close box in the upper left corner of the plot's title bar.

To move a plot to a new location on the screen, click on the title bar and drag the plot to the desired location.

To enlarge a plot, first click on it to make sure it is the active window. Then choose Display | Zoom In. To reduce the size of a plot, click on it to make it the active window, and then choose Display | Zoom Out.

3.4 Creating a New Dot Plot

To create a new dot plot:

1)     Choose Display | New 2D-Plot.
2)     A dialog box with a list of parameters will appear. Select the desired parameters for the X and Y axes.
3)     Click OK.

3.5 Painting a Population of Events

Painting allows the user to select a group of events on a dot plot and label them with an identifying color. Painting a population of events allows several types of further data analysis:

  • Events painted a particular color on one dot plot will be labeled by Paint-A-Gate with the same color on all other plots generated from that data set. This allows the user to define a population based on a pair of parameters, and then see how other parameters are distributed in that population.
  • Paint-A-Gate can display information such as the percentage of total events or the number of events represented by a painted population.
  • Painted populations can be gated (see Section 3.9), allowing further data analysis.
To paint a population of events:
1) Select the Paint menu, and choose a color: Red, Green, Blue, Yellow, Violet, Cyan, or White.
2) If the plot you wish to paint in is not the active window, make it active by clicking in it. You should see the cursor change from an arrow shape to a lasso shape.
3) Click and drag the cursor to encircle the events you wish to paint. The dots representing those events will change to the color you selected.
4) If you did not paint all the events you wanted on your first try, you may continue to paint with the same color until you choose another color from the Paint menu.

Note that Paint-A-Gate only allows you to paint on dot plots, not on histograms. However, events that have been painted on a dot plot will be displayed in the appropriate color(s) on a histogram plot.

3.6 Gating a Population of Events

Gating allows the user to select a subset of events from a data set, and have Paint-A-Gate treat them as if they were the entire data set. When the user has gated a set of events, non-gated events will not appear in plots or be included in the statistics calculated on the percentages bar or the Results Page.

Gating allows the user to further analyze a subset of the total population of events. Consider a user examining a data set collected on a mixture containing four distinct groups of events: FITC-labeled yeast, Tetrahymena which had ingested FITC-labeled yeast, Tetrahymena which had not ingested the yeast, and miscellaneous cellular debris. To determine what percentage of the Tetrahymena had ingested yeast, the user might begin by by painting the dataset to distinguish between Tetrahymena, yeast, and debris, as illustrated in Figure 3.

Figure 3. Plots of SSC vs. FSC (A) and SSC vs. FL1-H (B) for a dataset, with Tetrahymena painted red, yeast painted green, and debris in gray.

After painting, the user could gate the Tetrahymena events, causing Paint-A-Gate to ignore the yeast and debris events, as shown in Figure 4.

Figure 4. The data set from Figure 3, after gating the Tetrahymena events. The yeast and debris events have disappeared, and the Tetrahymena events have reverted to gray.
To complete the analysis, the user could repaint the Tetrahymena events as shown in Figure 5. All percentages and statistics calculated after gating are calculated on the gated population only. Thus, in this case, the green number in the Paint-A-Gate percentages bar after the repainting would represent the fraction of total Tetrahymena that had ingested yeast.

Figure 5. After gating, the Tetrahymena events have been repainted. Tetrahymena that have not ingested yeast are painted red; those that have ingested yeast are painted green.
To gate a population of events:

1) First, paint the population you wish to gate, as described above under Painting a Population of Events (Section 3.6).
2) Choose Manipulate | Gate Events, and then choose the color corresponding to the events you wish to gate.
3) All current plots will be re-drawn with only the gated events shown. The gated events will revert to gray color.

To undo the gating and display all events in the data set once more, select Manipulate | Show All Events.

3.7 Zapping Painted Events

Undoing the color labeling of a group of events and returning them to the default gray color is called "zapping" in Paint-A-Gate.
To remove the color from a group of painted events, select Manipulate | Exact Zap, and choose the color you wish to remove. The dots of that color will revert back to gray.

3.8 Displaying Data for Painted Events

Paint-A-Gate calculates statistics on painted populations. The percentages of the total population represented by painted events are displayed on a black bar just beneath the menu bar, shown in Figure 6. As the user paints events in red, for example, the red number on the menu bar reflects the percentage of the total events represented by the red events.

Figure 6. The Paint-A-Gate percentages bar, showing percent of total population represented by painted events.
To show more statistics, including number of events and mean values of parameters for all painted events, choose Report | Show Results Page. A pop-up text window will open with these statistics listed.