Agilent Technologies NovoCyte Quanteon Operator's Manual page 86

Running Samples
To collect the data, first double-click the sample to be collected in the Experiment
Manager to make it the Active Sample. Then place the corresponding sample tube
into the sample holder and click Run button. Repeat the procedure until all the
compensation samples are collected.
After all the samples are collected, the NovoExpress software will automatically
generate an FSC/SSC density plot, a histogram and a series of two parameter
density plots 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 for each sample. In the FSC-H/SSC-H density plot, a gate
named Main will be automatically created for the main compensation beads or cell
populations and be applied to all rest plots. NovoExpress will also set a Bi-Range
gate for the histogram to separate the negative (Negative gate) and positive
(Positive gate) populations, as shown in Figure 52.
Figure 52. Data Analysis for PE Single-Stained Sample in Auto Compensation
Under normal circumstances, the automatically generated gates are appropriate.
However, due to the complexity of the sample, in some cases, the automatic
software gating algorithm may not be able to generate the optimal gates. Check
the Main, Negative, and Positive gates to see if they are properly set. When
necessary, adjust the gates manually to optimize the results.
If the single-stained sample does not have a negative population, be sure to always
NOTE
include an unstained sample for automatic compensation. Otherwise, the
NovoExpress software cannot conduct automatic compensation and an error
massage will be generated.
User may need manually adjust the gates when significant amount of debris or
NOTE
complex populations are observed for single-stained sample.
4 Automatic Calculation of Compensation Matrix
The NovoExpress software will automatically calculate the compensation matrix
based on the signals (Mean or Median) of the negative and positive populations.
The font color of Compensation under the Compensation Specimen node in the
Experiment Manager turns blue to indicate the compensation matrix has non-zero
86 NovoCyte Quanteon Flow Cytometer Operator's Guide