Visualizing Protein-Protein Interactions in βTC3 Cells

Transfected βTC3 cells were incubated in serum-free imaging medium [125 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, 2 mM glucose, and 0.1% bovine serum albumin (pH 7.4)] for 3 h before imaging. Images were collected using a Zeiss AxioObserver microscope equipped with a 20×, 0.75 NA Plan-Apochromat objective. For heterotransfer experiments, cells were excited with a 455 nm light-emitting diode and a T455LP filter cube (Zeiss). CFP-emitted light and YFP-emitted light were separated by a Dual-View instrument (Optical Insights) containing the CFP and YFP filter sets. For the homotransfer experiments, cells were excited using polarized 505 nm light and a 46 HE YFP filter cube. Parallelly (P) and perpendicularly (S) oriented emitted photons were separated by a Dual-View instrument (Optical Insights) containing the polarization filter set.^{18 (link)–20 (link)} Images were collected at 37 °C using a Zeiss incubation system with a water-cooled Hamamatsu C10600 Orca R2 camera. Curve fitting and statistical analysis were performed using GraphPad Prism.