Creating New Experiments

CytExpert software separates color compensation and sample acquisition experiments into separate files designated .xitc and .xit, respectively. The experimenter can choose which one to execute first.

Creating New Experiment (.xit)

1. Select New Experiment in the File menu, specify the file path and name the experiment or accept the default naming Exp_YYYMMDD-1 and select Save.
2. Select Set Channel from the Settings menu. Select the desired channel signals in the checkbox. Type in reagent name in the label column if desired.
3. Explore the tool bar buttons to create plots in the experiment workspace. Select the axis name on the plot to change which channel is displayed.
4. To create gates, use the gating control buttons on the toolbar or right click the plot and select the gate type required.
5. Select the heading area of the plot to assign the gates to the required plots.
6. Select the Population Hierarchy button from the toolbar to display the population hierarchy. You can change color and names of gates by double clicking the color or name in population hierarchy window.
7. Select the Statistics button for the toolbar to display statistics window. Right click the Statistics table and select Statistics Setting and check desired header, statistics and populations of interest.
8. Select the Acq. Setting button on the left side panel to open the Acq. Setting window and display cytometer gains and threshold settings. In the Gain tab, select Recommended in cases where you do not specify your own default parameter gain settings.
9. Import compensation matrix to apply compensation values to experiment if required by selecting Compensation Matrix from the Settings menu to bring up the compensation matrix table. Select Import from Library or Import and locate the compensation matrix file (.comp) to import. Select desired option for importing compensation matrix and gain settings. Refer to Importing and Exporting Compensation in Compensation module of the Cytoflex IFU for more information.

Sampling and Collecting Data

1. Place the sample tube in sample tube holder and select Run to load the sample. In Acquisition panel, select sample flow rate and type in desired acquisition settings for Events to Display, Events to Record, and Time to Record.
2. Adjust gain settings for the different channels in Gain tab in the Acq. Setting window or use the Gain Control button on the toolbar directly on the plot where the data appears during data collection. Raising the gain increases the signal. Lowering the gain decreases the signal.
   Note: Optimize the gain settings according to your own experimental goals. The recommended settings are only for reference.
3. Adjust the threshold settings if required to remove unnecessary signal noise by selecting the Threshold tab in the Acq. Setting window. Choose the channel for threshold setting and manually enter a value in Threshold tab. Alternatively, select the Threshold button from the tool bar and move the mouse to desired threshold position in the desired plot.
4. Select Record to save the data and wait for the saving process to finish. The sample tube holder returns to the loading position. Add additional tubes to Tube list if required, load tubes and record data for each tube until all data required has been collected.

Identify Statistics to be Exported with the Experiment

Statistics table can be generated by selecting the ‘Statistic’ icon from the Workspace tab.

1. Once the statistic table pops up, right click on the table to access the menu.
2. Select Statistics Settings and here choose the statistics, population, and the header you would like to display.
3. Select Apply to All Tubes at the bottom of the Statistic Setting window if you want to apply to all the samples. Select OK once done.
4. If you would like to export the statistics to a csv table, right click on the table and select Export All Samples to CSV File. Choose the directory and save the file.
5. You can open this file in Excel and furthermore save as an .xls file.

Data Analysis

1. Open an existing experiment (.xit)
   File Menu > Select FCS File, Open. Imported files now have a green circle.
2. Choose the analysis icon on left side of screen
3. Use plotting tools as needed to complete the analysis. Multi-data or overlay plots (pull-down menu). Select the diamond tool to fill overlays, or drag and drop files.

Refer to user manual instructions for data analysis for more detailed instructions for use.