FCS Express: Tips for iCyt ec800 users

FCS Express is fully compatible with data acquired on the iCyt ec800. This page contains tips for working with data acquired with the iCyt ec800 and applications examples of working with single file and plate based overlays. The resources below are particularly useful when scaling the axis of plots and working with 96-well microplates.

Please also see our resources for working with Spectral Data.

Quick Links to Topics

Exporting from the ec800 Analysis Software
Changing Axis Titles
Scaling Axis
Working with Plate Based Data - Heat Maps
Applications Examples and Tutorials - Working with Histogram Overlays

Exporting from the ec800 Analysis Software

From within the ec800 Analysis software:

  • Select the Runs tab, highlight the names of the files you would like to export, and select the Export button on the upper right-hand side of the screen (1 below).
  • Select Export Type: FCS 3.0  File from the dropdown menu (2 below)
  • Select the Full resolution instrument data radio button (3 below)
  • Using the navigate button on the right hand side of the File/Path (4 below), select a Directory, and Folder you would like the data to be exported to
  • Click the Export button (5 below). Note: Click on image below to enlarge.

 

Once your data has been exported from the ec800 Analysis Software it may be loaded into FCS Express and treated like any other FCS file. Please visit our tutorials web page for detailed instructions on how to insert plots, assign gates, and use the many other features available in FCS Express.

Notes: You may choose to export the data as Full resolution instrument data or 10-bit analyzed data (step 3 above).

  • Full resolution” saves the data as a 19 bit FCS 3.0 file.
  • 10 bit analyzed data” saves the data as a 10 bit FCS 3.0 file.

Full resolution data is always stored as uncompensated linear data. The compensation matrix from the ec800 will be automatically applied to the data, depending on the preferences set in FCS Express. However, you will always have the option of changing or creating your own compensation. In addition, with full resolution data you get the benefit of being able to zoom into different portions of the data, as shown in the Axis Scaling steps below.

For 10 bit data, the compensation, log, log transforms, and user applied digital gains are stored directly in the data and is not modifiable. The 10 bit data will have the advantage of being of smaller size, and likely you will not have to change the axis scaling.

Changing the Axis Titles

Depending on the instrument, and on the settings that were used during acquisition, the parameter names might be stored in the Name or Stain keyword of the parameter in FCS file. The ec800 uses the Name keyword.

In FCS Express you can choose which keyword you would like displayed before the plot is opened. You can do this from the File tab→Options. Then, click on Data Loading, and select the FCS File Options category.

Then, change the value of Keyword to Use to Parameter Name.

You can read more information about setting up axis labels in our manual:

Axis Scaling

Sometimes FCS files do not have to use the full range of an axis and this may result in plotted data looking as if it is compressed on one or both axis. FCS Express makes it easy to correct this through use of the Magnify Axis and Magnify Resolution features. To use these features simply follow the steps below.

1. Right click on the plot to reveal the drop down menu (Figure 1). The Magnify (Resolution) and Magnify (Axis) options are located near the bottom of the list.

 

2. Choose Magnify (Axis) (Figure 1).

 

Figure 1 - Choosing the Magnify (Resolution) or Magnify (Axis) options from the right click drop down menu.

3. Left click and hold down the mouse button on the plot you would like to magnify. A "+" mouse icon will appear and you can now draw a box around the area of the plot you would like to magnify (Figure 2).

Figure 2- Choosing the area to magnify by drawing a magnification box around the region to be magnified.

At this point you have magnified the axis of the plot but the resolution may not be ideal for the new scaling resulting in the data looking smeared or blocky as in figure 3 (right).

Figure 3 - Before (left) axis magnification and after (right) axis magnification. Notice how the resolution in the magnified plot is less than ideal.

Simply repeat steps 1 through 3 above with the Magnify (Resolution) option instead of Magnify (Axis). The resulting plot will then look ideal as in figure 4 (right).

Figure 4- Before (left) magnification of axis and resolution and after (right) magnification of axis and resolution.

96 Well Plate Analysis

Since experiments on the iCyt instruments may be performed using 96-well plates, there are often many data files per experiment. Panels is a feature in FCS Express Pro that allows users to quickly and easily change the data sets displayed within a user-defined layout. Our High-content add-on allows you to easily visualize 96 well plates in a heat map plot and allows use of our new heat map gates. Please visit our high content tutorial for detailed instructions of how to use these features.

iCyt ec800 data displayed using the High-content heat map add-on for FCS Express.

 

Loading Data as a Plate

Most data files contain the well that they were acquired from, but do not provide any information about the size of the plate they were acquired from. FCS Express requires the plate size information in order to display the heat map of the correct size.

Since the size of your plate is not included in the ec800 data file you will need to download the file below containing plate format templates. Please click on the link below to download the Plate Template.zip file. The folder contains templates for 6, 12, 24, 48, 96, and 384 well plates. After downloading, please extract the contents of the .zip file and place the appropriate plate template text file within the folder of you plate based data. The next time you open your data in FCS Express the template will be read and your data will be displayed in the format you defined.

Once the plate template is placed in the folder with your data you may insert a heat map by selecting Insert Heat Map from the Insert tab of the Ribbon and choosing FCS Plate folder from the Files of type in the Select a Data File window (See below).

iCyt EC800 Applications Examples and Tutorials

Working with Multiple File Overlays

FCS Express makes it easy to create multiple histogram overlays for your iCyt ec800 data. Use stacked histograms to quickly make sense of titration series and insert custom statistics or regression analysis to see how data responds to gating changes in real time. See for yourself by downloading the PDF version of the layout: Working with Multiple File Overlays PDF or by using the free FCS Express Reader: Sony Histogram Overlays.

Tutorial Download: Creating Basic Overlays with iCyt EC800 Data.

Working with Plate and well Based Overlays

FCS Express makes working with your iCyt ec800 plate based data easy. Use well gates to pool data, create histogram overlays for multiple wells and parameters, and quickly insert and view standard and custom statistics. See for yourself by downloading the PDF version of the layout: Working with Plate Based Multiple File Overlays PDF or by using the free FCS Express Reader: Sony Plate Based Overlays.

Tutorial Download: Creating Basic Overlays with Plate Based iCyt EC800 Data.

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