Agilent Technologies NovoCyte Quanteon Operator's Manual page 93

Running Samples
There are three methods to edit the spillover matrix coefficient. One is by clicking
the Quick Compensation icon and moving the slide bar on the corresponding
fluorescence channel density plot directly (Figure 61). Another method is by
clicking Home > Compensation Matrix to load the compensation editing window.
The third method is by right-clicking the Compensation under the corresponding
sample node in the Experiment Manager and selecting Spillover Matrix ... to load
the compensation editing window.
Figure 61. Quick Compensation
A quadrant gate helps to judge if the correct compensation has been achieved. For
example, in PE/FITC density plot, draw a quadrant plot to divide the plot into four
segments. Include the PE negative population in the bottom-left segment and the
PE positive population in the bottom-right segment. Use the quick compensation
sliding bar or adjust the coefficient in the spillover matrix to conduct the
compensation. The correct compensation is achieved when the Mean or Median
of the FITC signal for the PE positive population and PE negative population is the
same or is very similar. The coefficient in the spillover matrix directly indicates the
percentage of PE signal spillover into FITC channel. Click the corresponding
coefficient in the spillover matrix, the information will be shown in the bottom-left
of the spillover matrix indicating a certain percentage of one fluorescence signal
(fluorophore in the corresponding column) need to be subtracted from the other
fluorescence signal (fluorophore in the corresponding row), as shown in Figure 62.
In the same way, compensate the PE spillover into the PerCP and APC channels on
the PE/PerCP and PE/APC density plots. Compensation for PE signal is then
completed, and results are shown in Figure 63. The spillover matrix for PE single-
stained sample is shown in Figure 62.
NovoCyte Quanteon Flow Cytometer Operator's Guide 93