Figure 5.

Specific binding of DAPA to HaloTag receptors. HEK 293 cells expressing surface HaloTag (HT-TM-mCerulean3, cyan; Addgene: 145767) were labeled with 500 nM DAPA or MG-BTau anchor-less control, and unbound dye was washed away (6×). Binding of DAPA via hexCl ligand was confirmed by the addition of 1 μM FAP, dL5**, which binds to any captured MG and should show a red fluorescence signal on HaloTag expressing cells. Cells were imaged using the Zeiss 880 LSM using 633 and 405 nm excitation with laser powers of 55 and 25.7% respectively and are depicted with 20 μm scale bars. DAPA specifically labeled mCer3 positive cells, indicating specificity of the DAPA for labeling HT expressing cells and that the anchored fluorogen is readily activated upon interaction with the FAP.