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. 2022 Mar 20;12(6):e4366. doi: 10.21769/BioProtoc.4366

Figure 4. Quantification of ATTO488-PE membrane fluorescence. A.

Figure 4.

For giant unilamellar vesicle (GUV) analysis, an outer region of interest (ROI) is placed around the GUV membrane and its total ATTO488 fluorescence is determined (blue circle). Another ROI is defined within the GUV lumen to quantify background fluorescence (punctured blue circle). The average fluorescence per pixel of the lumenal ROI is determined and scaled to the pixel area of the outer ROI for background subtraction. B. Dithionite bleaching of ATTO488-PE fluorescence in five empty GUVs (eGUVs, coded in different shades of green). The arrowhead indicates dithionite addition. The red line is a monoexponential fit of the combined data (t½ = 0.56 min, span = 0.47). C. Dithionite bleaching of ATTO488-PE fluorescence in eleven individually tracked scramblase-containing GUVs (sGUVs, coded in different colors), normalized to the fluorescence value at t = 0 min. The arrowhead indicates dithionite addition. Four of the traces show complete loss of fluorescence by t = 3 min, four show complete loss of fluorescence by t = 6 min, and three still show detectable fluorescence at t = 12 min. The horizontal dashed line corresponds to 50% loss of fluorescence. Modified from Mathiassen et al. (2021) .