Manual Fluorescence Compensation - Agilent Technologies NovoCyte Quanteon Operator's Manual

Running Samples

Manual Fluorescence Compensation

In addition to automatic fluorescence compensation, users can manually adjust
the spillover matrix for fluorescence compensation. Similarly, a series of unstained
and single-stained samples are needed.
Manual compensation could be done using the following procedure:
Prepare samples for compensation
•
Run compensation samples
•
Edit the compensation matrix
•
Apply the compensation matrix
•
Prepare Samples for Compensation
Use the same method to prepare the unstained and single-stained samples as
described for automatic compensation.
Run Compensation Samples
Use the method described in the previous sections to collect the data for all the
compensation samples.
Edit Compensation Matrix
Create an FSC/SSC density plot and set a gate for the main population on the plot.
Then create a density plot with the fluorescence channel of the fluorophore used
to stain the sample as the X-axis parameter and each of the other fluorescence
channels as the Y-axis parameter. Apply the main population gate to all the
fluorescence channel density plots.
For example in this guide, for the PE single-stained compensation beads, a gate
named Main is created for the main beads' population on the FSC/SSC plot. Create
a PE/FITC, a PE/PerCP and a PE/APC density plot and apply the Main gate to all
the plots. For the PE/FITC density plot, adjust the spillover matrix coefficient to
subtract the PE signal from the FITC signal (i.e. FITC ‒ % PE) to compensate for
the PE spillover into the FITC channel. The criterion for correct compensation is to
make the Mean or Median value of the PE positive population and negative
population to match each other as closely as possible on the FITC channel. The
same method and principle apply to compensation of PE spillover into the PerCP
channel on the PE/PerCP plot and PE spillover into the APC channel on the
PE/APC plot. Similarly, for the FITC, PerCP and APC single-stained samples, follow
the same procedure to subtract the corresponding fluorescence spillover into the
other detection channels.
92 NovoCyte Quanteon Flow Cytometer Operator's Guide