6
Load a sample labeled only with a single fluorophore of interest and
masking stain (DAPI or Hoechst 33342) if flourescent masking is chosen.
Run the sample using the desired FlexiCyte
TM
protocol.
In Plot Manager, open a new scatter plot by clicking on the scatter plot
icon on the left-hand side of the data row.
Compensation 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 spill over into a neighboring
channel giving a false increase in signal in this channel. For example, green
fluorescent protein (GFP) mainly emits fluorescence in the green spectrum that
is usually detected in a green channel. However, GFP also emits a small amount
of fluorescence in the orange spectrum that can be weakly detected in an
orange channel. To compensate for this fluorescent spillover into neighboring
channels, a compensation factor can be set for each fluorophore and channel.
Guidelines for approximate compensation factors to be applied for commonly
used fluorophores is listed in the ‘User Adaptable Protocols’ application note,
but the exact value should be determined as described below.
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 axis
should be ‘Linear’ and ‘Intensity’. Click ‘OK’. Tip: change the y-axis range
to cover a larger, lower range so that the cell population is centred on the
y-axis.
If there is spillover of fluorescence between the channels, the cell
population will be placed diagonally across the graph area (Fig 5). If there
is no spillover, the cell population will be placed horizontally across the
graph area (Fig 6A).
Open the compensation matrix (Fig 6B) in Plot Manager by clicking on the
icon on the left-hand side of the data row.
