Experiment Setup

Creating New Experiments

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

Creating New Experiment (.xit)

  • Begin an experiment by choosing new Experiment from the CytExpert landing page or from the ‘File’ tab
  • Default naming for experiments is Exp_YYYYMMDD.xit.Select Save.
  • Select ‘Settings’ from the menu and then ‘Set Channel’ to choose and label the parameters
  • Import compensation by selecting ‘Compensation Calculation’ from the ‘Settings’ menu which will bring up the matrix table.Select Import from library or Import from the designated .comp files.
  • Generate plots for the experimental view, open statistics table and generate gating hierarchies
  • Explore the tool bar:histogram, plot (various), Multiple Histograms, Multiple plots, Statistics, Hierarchy, Gate tools, zoom, Scale, Gain, Threshold, Undo/redo
  • Use mouse cursor to draw gate.
  • Right click on labels to change to another parameter.
  • Assign gate using plot title.
  • Set acquisition stop conditions
  • Use scale and gain to move data on plots, right click on the plots, gates, and stats to modify attributes.
  • Use single or dual threshold to set discriminator

 

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.

Selecting a sample injection mode:

  1. Manual
  2. Semi-Automatic
  3. Plate (available if plate loader was purchased)

CytoFLEX can support up to 3 sample injection modes as listed above.  Changing between sample injection modes requires user to physically change the sample probe or toggle the plate switch, change settings in CytExpert software, and then Cycle power on the CytoFLEX.

 

Open an Existing Experiment

  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.

 

 

Things to Remember

Max event rate is between 25,000 – 30,000 EPS;
Change from Default to High when event rate is >10,000 EPS.
Dead volume on U and V bottom plates is 10uL, flat bottom is 20uL.
.xit is experiment
.xitm is template
.xitc is compensation experiment