Single Cell D1ER FRET
D1ER stable HEK293 cells transfected with siRNA (~4–5 days) were imaged using an inverted Olympus IX81 fluorescence microscope. The cells were imaged in HBSS (Invitrogen # 14025), with 20 mM HEPES (pH 7.4), 1mM CaCl2 or calcium and magnesium free HBSS (Invitrogen Cat # 14175) with 20 mM Hepes, 0.5mM EGTA and 1mM MgCl2 at room temperature. Cells were alternatively excited with 340 and 380 nm light (Lambda DG-4; Sutter Instrument Co.) for 100–200 ms, and the emission >510 nm was captured using a CCD camera (Orca-ER; Hamamatsu Photonics) and analyzed with Slidebook (Intelligent Imaging Innovations). After background subtraction, the ratio of the 340- and 380-nm excited images were collected.