Determination of the oligomeric size distribution of FGF2-GFP by single particle brightness analysis. (A and B) Representative histograms of the fluorescence intensity distribution from individual experiments. CHO-K1 WT and CHO-745 mutant cells expressing FGF2-sfGFP at low levels were imaged to allow for single particle detection. Single FGF2-sfGFP particles localizing at the plasma membrane were identified, and their fluorescence intensity was measured and plotted. Three independent experiments were conducted with each of them including the analysis of at least 30 cells. (C) Comparison of the oligomeric state of FGF2-sfGFP at the inner plasma membrane leaflet of CHO-K1 and CHO-745 cells. The plotted data represent mean values (± SD based on three independent experiments). (D) Calibration curve of sfGFP oligomeric standards. Correlation between the mean fluorescence intensity and the sfGFP copy number for standard oligomers with different subunit numbers. Mean values and SDs from two independent replicates are shown. The red line represents the linear fitting of the data (r² = 0.997). The slope of the curve corresponds to the theoretical fluorescence intensity value of a single sfGFP molecule. (E) Intensity distribution of FGF2-mGFP at the plasma membrane of CHO-745 cells from a representative experiment. Approximately 3,500 particles were analyzed. The resulting histograms were fitted with a sum of Gaussians to estimate the occurrence of monomers (orange), dimers (green), trimers (yellow), and tetramers (magenta). The area under each curve was used to calculate the percentage of occurrence of each oligomeric species. (F) Percentage of FGF2-mGFP monomers, dimers, trimers, and tetramers calculated after labeling correction from the averaged distributions of species from two different experiments. (G and H) Fluorescence intensity plots of two representative individual FGF2-mGFP particles in CHO-K1 and CHO-745 cells showing two sequential photobleaching steps. (I) A model proposing a two-step process of FGF2 membrane translocation with a slow component (FGF2 oligomerization and membrane insertion) and a subsequent fast component (FGF2 capturing at the outer leaflet mediated by heparan sulfates).