Figure 3. Single-cell lipid mixing assay.

(a) Schematic of the approach. NDs containing 1% each of DiI and DiD lipid labels were pre-incubated with cells at 4 °C for 30 min to allow docking, but no fusion, with tCells. After rinsing twice with ice cold PBS to remove free NDs, PBS pre-heated to 37 °C was added and image acquisition was started and continued for 20 min using a spinning disc confocal microscope equipped with a temperature controlled stage set to 37 °C. For each image cycle, one frame recorded DiI fluorescence excited at 561 nm and the subsequent frame recorded DiD fluorescence excited at 647 nm. DiI fluorescence reports lipid mixing; upon fusion DiI and DiD are diluted in the plasma membrane and DiI is no longer quenched by DiD. At the labelling density used, DiD is not significantly self-quenched, so the DiD signal is proportional to the initial density of docked NDs. The ratio R of DiI-to-DiD fluorescence normalizes fusion signals for variations in docked ND density, temperature-induced fluorescence changes, and other instrumental and environmental factors. (b) Changes in DiI-to-DiD fluorescence ratio (ΔR), relative to the initial ratio (R_o), for SNARE-free NDs (eND), or NDs loaded with wild-type (vND) or docking-competent, fusion-incompetent VAMP2-4X (v4xND). 11, 8, and 6 dishes were analysed for vND, v4xND and eND conditions, respectively.