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Figure 2 A) Cy5 mRNA or FITC mRNA is encapsulated separately into single-lipoplexes, and the desired R*/G* ratios are obtained by mixing single-R*- and single-G*-lipoplexes at an R*/G* ratio of 100/0%, 75/25%, 50/50%, 25/75%, and 0/100% w/w. B,C) Single-lipoplexes mixtures that contain Cy5 luciferase mRNA single-lipoplexes and FITC luciferase mRNA single-lipoplexes at an R*/G* ratio of 100/0%, 75/25%, 50/50%, 25/75%, and 0/100% w/w were incubated with HeLa cells to examine the cellular uptake. B) After 4 h incubation, cells were rinsed and left to grow for another 20 h in fresh culture medium before flow cytometry measurements. C) After 24 h incubation (no washing step) cells were examined by flow cytometry. D,E) Single-lipoplexes mixtures that contain mCherry mRNA single-lipoplexes and eGFP mRNA single-lipoplexes at an R/G ratio of 100/0%, 75/25%, 50/50%, 25/75%, and 0/100% were incubated with HeLa cells for (D) 4 h (followed by a washing step) or (E) 24 h (no washing step) and protein expression was determined at 24 h (E1, E2) by flow cytometry measurements and (E3) by confocal microscopy. (B1, C1, D1, E1) Percentage of co-positive cells, (B2, C2, D2, E2) correlation between red and green fluorescence as derived from flow cytometry dot plots. Scale bar: 100 µm. All the data was averaged from three independent experiments, with three replicates per repeat (n = 9). The total mRNA amount was kept constant at 0.2 µg/well.