FCS Express: Tips For Beckman Coulter Users

Listmode files

Beckman Coulter listmode files acquired on the FC 500, Gallios™, and Navios™ instruments actually contains two datasets inside. The first data set is 10-bit FCS 2.0 data. This is the dataset that you are used to seeing. The second dataset is 20-bit FCS 3.0 data. Users can decide to analyze one or both datasets in FCS Express.

You can choose which dataset to use in the FCS File Options category of FCS Express User Options. To have access to both datasets, you will need to check the box next to Allow Multiple Datasets. Then, when you select the listmode file, you will be given the option to choose which dataset to load.

Leave the box unchecked to always select a single dataset. Enter the data set you wish to use (one (1) for the FCS 2.0 dataset or two (2) for FCS 3.0 dataset) in the Default Dataset to use edit box.

Baseline Offsets

If you have a Beckman Coulter flow cytometer, you probably already know that they have a feature called "baselines offset" that allows you to visually enhance your data display. FCS Express also has this functionality and here is a short video to show you some tips for handling this type of display.

Using Baseline Offsets

Converting the Gallios Time Parameter to Real Time

The Gallios allows you to define Acquisition and Elapsed Time settings. For more details on the Elapsed Time setting please see our elapsed time knowledgebase article. FCS Express can be used to convert the Time parameter in the Gallios file to real time through the use of the parameter math feature. To convert the Time parameter stored by the Gallios to real time please use the following formulas with parameter math:

For the FCS 2.0 dataset For the FCS 3.0 dataset

If P14 is the time parameter:

New Parameter=P14*(Elapsed time/1024)

If P14 is the time parameter:

New Parameter=P14*(Elapsed time/1048576)

 

The values for the Elapsed Time can be found in the Gallios Cytometer Control dialog or the Set Times dialog as seen below.

If you want to see what Elapsed Time was used for your data file, you can look at the @PnX keyword in the data file, where n is the time parameter in that data file. The @PnX keyword is only located in the FCS 2.0 dataset of the Gallios file.

Displaying Summit data in FCS Express as it appears in the Summit Software

FCS data in the Summit software is displayed on a 0-256 linear or 0-100,000 logarithmic scale. However, the data is stored in the data file as 32 bit data with a range of 0-4,294,967,295. When the data is viewed in FCS Express the 32 bit native values are displayed on the axis by default and used for subsequent calculations such as means and medians which may appear different than it originally looked in Summit Software. The instructions below may be followed if you are noticing a problem with the way the data is displayed.

How to rescale Summit data (after 6.004.0034 release)

In the newest version of FCS Express, an option is available for users to scale fluorescence parameters on a 5 log decade scale so the data is displayed identically to Summit Software. To acess this feature, you will need to open your FCS Express User Options by selecting the File tab and selecting FCS Express User Options from the drop down menu. Once the FCS Express User Options window is available, FCS File Options can be selected fom the expanded view of the Data Loading option. The Scale Summit fluorescence parameters box can be checked. This will update the user preferences to scale Summit data in 5 log decades anytime a file is loaded. This preference change will only need to be done once for each user. Below is an example of the difference of the default scaling and the updated scaling.

FCS Express User Options

 

Native Summit Data
Newly Scaled Summit Data

How to rescale Summit data in older versions of FCS Express