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A.D. Albert, M.A. deLivron, M. Katragadda, M. Lynes; Probing Rod Outer Segment Disk Membranes Using Flow Cyotmetry . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1265.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Flow cytometry is a powerful technique that can be used to investigate surface components and structural features of a cell population, as well as to sort cells as a function of size. This technique allows cells and organelles to be distinguished based on a fluorescent signal as they pass in a stream of single particles through a laser beam. The objective of this work is to establish flow cytometry as a means to investigate disk membranes. This represents, to our knowledge, the first time flow cytometry has been applied to disk membranes. Methods: Disk membranes were isolated from bovine rod outer segments by ficol flotation. Flow cytometry was used to measure changes in disk size and fluorescence before and after bleaching. Oxidative damage has been linked to retinal degenerations. Therefore effects of oxidation were investigated by exposing disks to hydrogen peroxide prior to employing flow cytometry. Caveolin, the sphingolipid, ganglioside GM1, and cholesterol (components linked to membrane rafts) were separately labeled with fluorescent antibodies. Flow cytometry was used to examine the distribution of these molecules among the disks. Results: Bleaching caused an apparent decrease in FL1 fluorescence and in the size of disks. Regeneration with 9–cis retinal, largely reversed these changes. After exposure to peroxide, there was also an apparent decrease in size and FL1 fluorescence. Flow cytometry data from disks labeled using fluorescent antibodies indicate that caveolin and the sphingolipid, ganglioside GM1, are inhomogeneously distributed within sub–populations of the disks. Furthermore, flow cytometry data on disks labeled with anti–cholesterol anti–body are consistent with earlier work from this laboratory showing cholesterol is inhomogeneously distributed among the disks. Conclusions: Our data show that flow cytometry can distinguish disk membranes and that the disks can be tagged for specific membrane components. Flow cytometry offers a new means to examine osmotically intact isolated disks from rod outer segments and how they change during spatial displacement.
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