Cytek Biosciences Aurora User Manual page 49

Select Universal Negative for single-stained FCS files that do not contain a negative population.
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In the bottom left of the screen, check whether Auto Fluorescence will be used as a fluorescent
tag.
Select Refresh Plots to display the data in the FSC vs SSC plot, peak emission channel histogram,
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and spectrum plots. The positive and negative populations need to be identified through the
appropriate placement of the existing gates.
a. Move the polygon gate in the FSC vs SSC plot to include the singlet population. To apply this
gate placement to all FSC vs SSC plots, hold the Ctrl key while moving the gate.
b. Move the interval gate on the spectrum plot to select the channel that exhibits the brightest
fluorescence intensity. This channel is the peak emission channel for the fluorescent tag.
c. Move the interval gate in the histogram for the peak channel labeled Positive to include the
positively stained population. Move the interval gate in the histogram for the peak channel
labeled Negative to include the negative population.
Chapter 6: Advanced Unmixing 49