How to do compensation in Flexicyte


Although fluorophores are designated a particular color, they emit light over a range of wavelengths. The color associated with a particular fluorophore refers to the wavelength where it has maximum fluorescence, however the wavelengths where it has weaker emission may spillover into a neighboring channel giving a false increase in signal in this channel. Therefore, you need to do compensation if you want to measure two or more fluorophores at the same time on the NC3000 using Flexicyte. For example, AlexaFluor488 is detected in the green channel in the NC-3000™, however the emission from AlexaFluor488 can be weakly detected in the other channel as well. To compensate for this fluorescent spillover into neighboring channels, a compensation factor is set for each fluorophore and channel.

How to check the spectrum of your fluorophores

A great tool for checking the spectrum of your fluorophores is the “Fluorescence SpectraViewer” from Thermo Fisher. Furthermore, you can display your light sources and their extinction and emission filters as well. This will help you to decide if compensation is necessary or not.

How to use the compensation window

The Compensation Window is used to compensate for spectral overlap of fluorophores into neighbouring channels.For each channel it is possible to enter a subtraction percentage value to be used when compensating cell parameter values for spectral overlap. Click in one of the edit boxes to select it. A selected edit box is marked by red font and a description what channel spillover to what channel it sets is written below the slider. When an edit box is selected the value can be edited writing a new value and clicking enter or Preview. Another option is to use the slider by moving it with the mouse or with the arrow keys. When the correct compensation factor is ascertained click ‘Apply and Close’.

How to do the actual compensation – easiest way

The easiest way to do compensation would be to use the values listed in the table in the Flexicyte protocol. But those numbers are only approximate numbers and are not exact. Meaning, when it is not possible to have some single stained cell samples for each fluorophore ready to do the correct compensation and those fluorophores are listed in that table, use those numbers. But otherwise the compensation should always be done using single stained cell samples (see below). If you want to use those values from the table, enter them in the compensation window and apply this using the master row function to all rows. Now you even can generate a new protocol for the customer with the default settings.

How to do the compensation – correct way

Sample preparation

Doing compensation is time-consuming, but it is worth it! As an example, we will use three fluorophores,AlexaFluor488 and AlexaFluor647 and PI for detecting the dead cells. For masking we will take Hoechst 33342 for all cell samples. For the compensation you need to ask the customer to prepare different cell samples: Basically, this would be the protocol for the staining:

  • Harvest your cells and get them in cell single suspension. 
  • Count your cells and prepare them for 2E6 – 5E6 cells/ml in PBS/FACS buffer. As you only need to use approx. 50µl/slide, you may scale down to approx. 100-200µl, if you are running out of cells.
  • Stain them accordingly to your specific staining protocol. Note: As the NC3000 is not that sensitive than a flow cytometer, start with a high antibody concentration. You need these samples (if you have a positive control sample, please include it):
    • Unstained
    • AlexaFluor488
    • AlexaFluor647
    • Only PI (stain it directly before measuring using PI)
    • AlexaFluor488 + AlexaFluor647 + PI (stain it directly before measuring using PI)
  • Stain all cell samples with Hoechst 33342 for masking (10µg/ml for 15min at 37°C). If your Alexa antibodies are temperature sensitive, try it for 30min at 4°C.
  • Stain sample d and e with PI directly before measuring using 2µg/ml.
  • Load the A2 slides and run the samples using the adapted protocol on the NC3000.


After running the cell samples, you can start with the compensation. 

  1. Open all samples using the plot manager. 
  2. In Plot Manager, open a new scatter plot by clicking on the scatter plot icon on the left-hand side of the data row. 
  3. Double-click the small scatter plot to open the large scatter plot in editing mode. Change the x-axis to the colour of the fluorophore and the y-axis to the colour of the neighbouring channel. The axis settings for both axes should be ‘Linear’ and ‘Intensity’. Click ‘OK’. Tip: Change the y-axis range so that the cell population is centered on the y-axis. 
  4. If there is spillover of fluorescence between the channels the cell population will be placed diagonally across the graph area. If there is no spillover, the cell population will be placed horizontally across the graph area. 
  5. Open the compensation matrix in Plot Manager by clicking on the icon on the left-hand side of the data row. 
  6. Set the compensation factor in the matrix box associated with the two channels of interest, so that cell population is spread horizontally across the graph area. When the correct compensation factor is ascertained click ‘Apply and Close’. 
  7. Use the master row function to assume it for the other rows.
  8. Repeat steps 2-7 for each individual fluorophore. 
  9. If multiple samples have been analyzed, enter the row number of the sample with the corrected compensation factor into the ‘Master Row’ box situated in the top left-hand corner of Plot Manager and click ‘Apply’. This will replicate the compensation in all other samples selected in Plot Manager. 
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