Advanced Threshold Setting
When the different populations are not well separated, automatic thresholding fails and must be manually adjusted. The threshold is usually set using horizontal or vertical bars.
When a single fluorophore assay is used, the 1D dot-plot enables manual threshold setting
When two or more fluorescent assays are used, it is advised to visualize the 2D dot-plot of the results to verify if both thresholds are correctly set. Furthermore, the visualization using 2D dot-plot can be useful to understand how populations are distributed.
Manually adjust the threshold
Finally, if you need to manually adjust the threshold, do not forget to have a look at the wells containing your positive and negative controls included in each experiment. They provide information that can be useful for threshold setting.
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