Figure 3. Evaluation of dasatinib-BODIPY and saracatinib-BODIPY as probes for flow cytometry.

Src-mCherry expressing Huh7 cells were treated with DMSO (negative control) or with 100 nM saracatinib-BODIPY, dasatinib-BODIPY, or BI-BODIPY. (A) mCherry fluorescence profile versus forward scatter showing the presence of a distinct high fluorescence population (mCherry^hi) for each compound. This population was gated for further analysis. (B–D) For competition experiments, cells were labeled with 100 nM saracatinib-BODIPY, dasatinib-BODIPY, or BI-BODIPY in the presence or absence of 10 μM free saracatinib or dasatinib as competitor as indicated. Histogram plots for the BODIPY fluorescence intensity are shown. Unique peak profiles for saracatinib-BODIPY, dasatinib-BODIPY, and BI-BODIPY suggest that staining reflects binding of these probes to specific targets dictated by the kinase-targeting group and not by generic interactions of the fluorophore. (B) Competition with unlabeled saracatinib does not significantly reduce the fluorescence intensity of saracatinib-BODIPY. In contrast, unlabeled dasatinib significantly reduces the fluorescence intensity of (C) dasatinib-BODIPY but not (D) BI-BODIPY, suggesting that this competition is specific. Data are representative of four independent replicates.