Compensation



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

  1. If a new compensation is needed, generate as follows:
    1. Prepare all necessary unstained and single color controls using compensation beads or cells.
    2. .CytoFLEX new compensationSelect New Compensation in the File menu, name file and click Save
    3. Select the channels requiring compensation and then select Sample Type of the prepared control tubes. Unchecked tubes not needed for the experiment. Select OK.
      Note: Label and Lot No. information can be entered and retained in Compensation Library if desired.
      CytoFlex Compensation Library
      CytExpert automatically creates a list of matching empty tubes in the Tube panel as well as all the plots needed to gate the positive and negative populations.
      CytoFLEX Tube Panel
    4. Load the corresponding unstained or single color sample fluorospheres or cells and select run in the Acquisition panel.
    5. If necessary, adjust the scatter gate so that it encloses the desired population. Adjust the slider scale on fluorescence plot so the negative and positive peaks appear in a suitable position. Move the positive and negative gates to enclose the corresponding peaks.
    6. Once the gates are in satisfactory locations, select record.
    7. Repeat steps d-f for all tubes.
    8. After collecting data for all necessary compensation samples, generate the compensation matrix by selecting Compensation Calculation from toolbar or settings menu.
      CytoFLEX Compensation Calculation
    9. The Compensation Matrix window appears displaying the compensation values. Select Save As to export the matrix as a .comp file and specify file path. Select Save to Compensation Library to save the single color compensation values to the compensation library. Select OK and select Close.
    10. To save or overwrite the compensation matrix to the library, Open compensation experiment, run compensation controls. Once completed, calculate compensation. Then save the comp library using designated button under the compensation matrix (pop-up). “Save as Comp Matrix”, “Save as Comp Library”. Choose a name.
      Quick Tip: To apply compensation retroactively (starting with an experiment first, then applying compensation), open an experiment, run your samples. Right-click on sample, a box appears “Apply Compensation”. Next to your tube, there is a clear checkered icon/box (very left), right click on icon to apply compensation matrix. From there, choose your matrix. Then, apply compensation to all tubes.