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Materials, Methods & Technologies, Volume 10, 2016

A RAPID PLATFORM TO GENERATE LIPOFUSCIN AND SCREEN THERAPEUTIC DRUGS FOR EFFICACY IN LIPOFUSCIN REMOVAL
Jason Gaspar, Jacques Mathieu, Pedro J. J. Alvarez
Pages: 1-9
Published: 9 Feb 2016
Views: 3549
Downloads: 857
Abstract: Lipofuscin is a brown-yellow, autofluorescent polymeric material that accumulates in a ceroid manner within postmitotic cells during aging. Lipofuscin accumulation impairs proteosome and lysosome pathways critical to cell health and homeostasis. Therefore, the ability to quickly generate lipofuscin in vitro, and identify drugs that mitigate the accumulation or clear lipofuscin would be of great benefit to aging research. Here, we present a platform to quickly create lipofuscin-loaded (but otherwise healthy) cells and screen drugs for efficacy in lipofuscin removal. The combination of leupeptin, iron (III) chloride and hydrogen peroxide generates significant amounts of lipofuscin within cells while eliminating the need for a 40% hyperoxic chamber. Alternative methods which load fibroblasts with “artificial” lipofuscin obtained via UV-peroxidation of mitochondrial fragments are much more labor-intensive. This method is faster (≤10 days) than most protocols to generate lipofuscin and assess its removal, which typically require 2 to 4 weeks or longer to complete.
Keywords: lipofuscin, flow cytometry, fenton chemistry, autofluorescence, fluorescent microscopy
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