Journal: Soft Matter

Article Title: Quantification of cholesterol incorporation in giant unilamellar vesicles produced by a modified cDICE method

doi: 10.1039/d5sm01124h

Figure Lengend Snippet: NR12A generalized polarization (GP) as a reporter of cholesterol incorporation in DOPC GUVs. (a) Schematic representation of the change in GP of NR12A in DOPC membranes upon MβCD-driven delivery of cholesterol in the outer medium. (b) Representative color-coded GP images generated from confocal images at the equatorial plane of eDICE GUVs at different cholesterol (MβCD–CL) concentrations. Scale bars indicate 10 µm. (c) GP measured in eDICE GUVs at the different conditions. Box percentile 5–95, line in box is the mean and whiskers the SD. Each data point represents the average GP value of one individual GUV (we measured at least 50 GUVs per condition from at least 3 different replicates). Statistical significance was tested using a non-parametric Kruskal–Wallis test (** p < 0.01 and *** p < 0.001).

Article Snippet: Flipper-TR (SC020) from Spirochrome AG and MemGlow NR12A (MG07) from Cytoskeleton, Inc. were supplied by Tebu Bio.

Techniques: Generated

Journal: Soft Matter

Article Title: Quantification of cholesterol incorporation in giant unilamellar vesicles produced by a modified cDICE method

doi: 10.1039/d5sm01124h

Figure Lengend Snippet: Calibration of fluorescence lifetime and GP measurements of the cholesterol content of GUVs. (a) Mean GP of DOPC GUVs containing different proportions of cholesterol prepared by gel swelling. The dashed line with shaded outline represents the linear regression fit ( y = a + bx ; a = −0.542 ± 0.016 and b = 0.010 ± 0.001). (b) Mean intensity weighted lifetimes of DOPC GUVs and different proportions of cholesterol prepared by gel swelling. The dashed line represents the linear regression fit ( y = a + bx ; a = 2.925 ± 0.020 and b = 0.037 ± 0.002). In both panels a and b, dots and whiskers represent the mean ± SD (with at least 30 GUVs from 3 different replicates measured per lipid composition) and the shadowed area is the 95% confidence interval of the fit). (c) Cholesterol percentage in the membrane of eDICE GUVs as a function of the MβCD–CL concentration in the outer medium estimated from the calibration curves from gel swollen vesicles, measured by NR12A spectral imaging (based on the data in ) and Flipper-TR fluorescence lifetime (based on the data in ). Statistical analysis (non-parametric Kruskal–Wallis test) confirms that there is no significant difference (n.s.) in the results obtained from each method. Points represent the cholesterol molar percentage estimated for each single GUV. Box percentile 5–95, line in box is the mean and whiskers the propagated errors.

Article Snippet: Flipper-TR (SC020) from Spirochrome AG and MemGlow NR12A (MG07) from Cytoskeleton, Inc. were supplied by Tebu Bio.

Techniques: Fluorescence, Membrane, Concentration Assay, Imaging

Journal: Soft Matter

Article Title: Quantification of cholesterol incorporation in giant unilamellar vesicles produced by a modified cDICE method

doi: 10.1039/d5sm01124h

Figure Lengend Snippet: NR12A GP measurements of cholesterol incorporation for eDICE GUVs made of binary lipid mixtures. (a) Representative color-coded GP images of DOPC : PC(18 : 0–14 : 0) (60 : 40 mol%) GUVs at different cholesterol (MβCD–CL) concentrations in the outer medium. (b) GP values of DOPC : PC(18 : 0–14 : 0) GUVs as a function of MβCD–CL concentration. (c) Representative color-coded GP images of DOPC : DMPC (6 : 4 mol%) GUVs at different cholesterol (MβCD–CL) concentrations in the outer medium. (d) GP values of DOPC:DMPC GUVs as a function of MβCD–CL concentration. Scale bars in panels a and c indicate 10 µm. In panels b and d, box represents percentile 5–95, line in box is the mean and whiskers the SD. Each data point represent the average GP value of one individual GUV (we measured at least 50 GUVs per condition from at least 3 different replicates). Statistical significance was tested using a non-parametric Kruskal–Wallis test (** p < 0.01 and *** p < 0.001).

Article Snippet: Flipper-TR (SC020) from Spirochrome AG and MemGlow NR12A (MG07) from Cytoskeleton, Inc. were supplied by Tebu Bio.

Techniques: Concentration Assay