(a) Schematic of the assay. (b) NLPs co-labeled with one mole % DiI and DiD were incubated with tCells for 30 min at 4°C, a temperature that allows docking but not fusion. Cells were then rinsed with cold PBS to remove free NLPs, and PBS pre-warmed to 37°C was added. Imaging of DiI and DiD fluoresce started shortly after the dish was mounted onto a confocal microscope stage held at 37°C. For each imaging cycle, we sequentially acquired DiI and DiD fluorescence (λex =561 nm and 647 nm for DiI and DiD, respectively). We quantified cell membrane DiI and DiD fluorescence and calculated the ratio of these two intensities, R. DiI fluorescence reports lipid mixing, while the DiD fluorescence is proportional to the amount of docked NLPs per cell. Thus, the ratio R normalizes the lipid-mixing signal to the amount of docked NLPs. Averages of 69, 73, and 47 regions of interest (± S.E.M.) from 7, 7, and 3 dishes are shown for NLPs loaded with ~eight copies of VAMP2 (vNLP8), for NLPs loaded with ~eight copies of the VAMP2-4x mutant (which are docking-competent but fusion incompetent – v4xNLP8), and for empty NLPs (eNLPs), respectively. (c,d) Confocal imaging after 15 min incubation and washing of NLPs with tCells at 37°C. (c) DiD fluorescence reflects the amount of docked NLPs per cell. NLPs reconstituted with ~eight copies of VAMP2-4X (v4xNLP8) docked with the same efficiency as wild-type VAMP2 NLPs bearing the same SNARE copy number (vNLP8). (d) DiI/DiD fluorescence ratio (R) reports lipid mixing normalized to the amount of docked NLPs per cell. Despite efficient docking, v4xNLP8 did not induce any lipid mixing. eNLP, empty (SNARE-free) NLPs. For (c) and (d), 6, 10, and 11 dishes were measured and 41, 66, and 63 regions of interest analysed for eNLP, v4xNLP8, and vNLP8, respectively.