Compensation window – ChemoMetec NC-3000 User Manual
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991-3003 Rev. 1.0
August 2013
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Gate configuration:
Disable: This option disables gating for the selected gate.
Include: This option results in display of all events inside the selected gate.
Exclude: This option results in display of all events outside the selected gate.
Union: This option is available when two gates have been set to either Include or Exclude.
Selecting this option results in display of the sum of all events displayed for the two gates
(included or excluded).
Intersection: This option is available when two gates have been set to either Include or Exclude.
Selecting this option results in display of the events displayed for both of the two gates
(included or excluded).
Compensation Window
The Compensation Window is used to compensate for spectral overlap of fluorophores into neighboring
channels.
For each channel it is possible to enter a subtraction percentage value to be used when compensating cell
parameter values for spectral overlap.
Even though 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. For example, green fluorescent protein (GFP) is
detected in the green channel in the NC-3000™, however the emission from GFP can be weakly detected in
the orange channel. To compensate for this fluorescent spillover into neighboring channels, a
compensation factor is set for each fluorophore and channel. Guidelines for approximate compensation
factors to be applied for commonly used fluorophores is listed in the FlexiCyte™ application note but the
exact value should be determined as described below.
1. Load a sample labeled with a single fluorophore of interest and the blue DNA masking fluorophore
i.e. DAPI or Hoechst 33342 into the NC-3000™.
2. Run the sample using the FlexiCyte™ protocol.