90 BD FACSCalibur Instructions for Use
Optimizing Settings
Before data is collected, optimize instrument settings as follows:
The following control samples are recommended for 4-color optimization:
• isotype control
• compensation controls for each fluorophore in the assay (ie, FITC, PE,
PerCP, and APC controls)
Adjusting FSC, SSC, and FSC Threshold
Adjust the FSC Amp Gain and SSC Voltage to appropriately display the scatter
properties of the sample and place the population of interest (eg, lymphocytes) on
scale. Adjust the FSC Threshold to exclude unwanted debris.
For example, the following plots show cells before and after optimization of FSC
and SSC settings. Usually, Calib File settings need minimal adjustment to place
lymphocytes on scale.
Figure 5-1 FSC/SSC optimization
Adjust FSC and
SSC detectors.
Adjust FSC
threshold.
Gate population
of interest.
Adjust fluorescence
detectors.
Adjust fluorescence
compensation.
population of
interest
unadjusted adjusted
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