Figure 4.
Normalization of the Nef-BiFC signal to mRFP expression improves assay performance. Transfected 293T cells expressing the wild-type Nef-BiFC protein pair (WT2A) or the dimerization-defective control (4D2A) were imaged on the ArrayScan II platform, and the resulting data were analyzed in three different ways: (A) number of Nef-BiFC-positive (BiFC+) cells within each mRFP-positive cell population; (B) overall Nef-BiFC fluorescence intensity within the mRFP-positive subpopulation; (C) Nef-BiFC fluorescence intensity normalized to mRFP fluorescence intensity within each mRFP-positive cell. The statistical parameters resulting from each analysis are summarized in the tables on the right. Note that analysis based on the BiFC/mRFP ratio minimized variability and maintained high assay performance (Z factor > 0.6).