Running Samples
78 NovoCyte Quanteon Flow Cytometer Operator's Guide
threshold can be based on any one of the parameters set under the Cytometer
Settings > Threshold panel. When two threshold conditions are set, data will be
collected only from particles with signal intensities meeting both thresholds.
The Storage Gate is used to filter out events outside the gate. All events above the
primary and secondary threshold will be recorded when Storage Gate is set to
Ungated. In the example in this guide, the primary threshold is set to FSC-H greater
than 100,000, the second threshold is left blank and the Storage Gate is set to
Ungated.
Figure 45. Set the Threshold
During data acquisition, the threshold can be adjusted after clicking the Adjust on
Plot tab. The threshold will be set to the new value when clicking the desired
position on the plot for the subsequent data acquisition.
For a new experiment, it might be difficult to determine how to set the threshold. In
this case, conduct a pre-run with threshold adjusted on the plot to determine where
the cell population of interest is in the FSC-H/SSC-H plot. The pre-run results should
serve as a good guide for setting the proper threshold for the next experiment. In
the example in this guide, the distribution of the leukocytes (lymphocytes,
monocytes, granulocytes) relative to the lysed red blood cells and debris should be
observed. Adjust the FSC-H threshold value until the majority of the lysed red blood
cells and debris are discarded, but the leukocytes are not affected. Set this
threshold in the Threshold panel for the real experiment.
For new experiments, a pre-run helps to optimize the cytometer settings and
determine the most appropriate experimental conditions, including the Stop
Condition, Flow Rate, and Threshold.
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