Agilent Technologies NovoCyte Quanteon, Operator'S Manual, Page: 83 / 148

Running Samples
NovoCyte Quanteon Flow Cytometer Operator's Guide 83
control sample and a series of single-stained samples. The compensation matrix
can be generated either from samples acquired on NovoCyte Quanteon instrument
or from data imported from FCS files. Please refer to
Automatic Fluorescence
Compensation from Imported FCS Files in this guide for instructions on generate
compensation matrix from imported FCS files. The automatically generated
compensation matrix can be applied to the multi-color stained samples to achieve
correct fluorescence compensation.
Standard Automatic Fluorescence Compensation Procedure
The procedure for standard automatic compensation is described in the following
steps:
•
Prepare samples for fluorescence compensation
•
Set up automatic fluorescence compensation parameters
•
Run samples for automatic fluorescence compensation
•
Automatic calculation of compensation matrix
•
Apply compensation matrix to experimental samples
1 Prepare samples for fluorescence compensation
Prepare a set of single-stained samples (labeled with only one fluorophore for
each sample). Single-stained sample can be generated from the same biological
sample as the experimental sample, or compensation beads may be used.
Compensation beads are microspheres with capturing agent on the surface. The
capturing agent on the surface of the compensation beads can react with
fluorescent-labeled antibodies used in the experiment. Each single-stained sample
should have a negative and a positive population. If there is no negative population
in the single-stained sample, an unstained sample needs to be prepared for correct
automatic compensation.
2 Set up Automatic Fluorescence Compensation Parameters
Click Home > Auto Compensation icon , or right-click the experiment file name
in the Experiment Manager and select New Auto Compensation ... (Figure 49).
The New Auto Compensation window will appear (Figure 50). In this window, you
can set the method and parameters for automatic compensation. Calculation of
the compensation matrix can be set based on either the
Height or the Area
parameter and based on comparison of the Mean or Median of the positive and
negative populations. Select Compensation Channels based on the prepared
Unstained sample and the single-stained samples accordingly.
For the example described in this guide, single-stained compensation beads with
FITC, PE, PerCP and APC will be used for automatic compensation. In