A bursting assay for Giant Unilamellar Vesicles containing gangliosides

We know that certain cell types are constantly exposed to flow, that these cells sense flow and that flow is essential for normal function in these cells. We want to study the effect that flow has on membrane proteins. Our lab creates phospholipid bilayers in the form of Giant Unilamellar Vesicles (GUV) in order to investigate the effect of flow on membrane proteins. We break the vesicles using a saline solution; the vesicles sink and rupture on the glass surface to form a flat sheet (splat), which we refer to as a supported lipid bilayer (SLB). We then apply a flow to the SLB to see its effect on membrane proteins.This work studied how to optimize the breaking conditions of GUV to use the SLB to study the effect of flow. In order to characterize splatting conditions, we took a movie of the vesicles breaking on a glass surface and developed a Python program to detect the vesicles in each frame of the movie to observe the properties of the splatting. We found that as expected, there was a decrease in vesicle count through time, increasing gangliosides did not have an observable effect on splatting, and cleaning the slides with plasma did not make the vesicles splat faster.