Development of a novel fixation method for mitochondria

Abstract

At biological pH, isolated mitochondria carry a negative charge on their surface as a result of the chemical composition of the ionizable groups on their surface. Capillary Electrophoresis with Laser Induced Fluorescence Detection (CE-LIF) has been shown to separate and detect fluorescently labeled individual mitochondria based on their electrophoretic properties. This technology has been used to provide insights into the role of mitochondria in aging and disease. A problem exists, however, with the stability of isolated mitochondria. CE-LIF analysis of mitochondria must be done within a few hours after the sample preparation. Otherwise, the electrophoretic properties of a mitochondrial preparation begin to change. This prevents collaboration among laboratories and limits the number of tests that can be done on a given mitochondrial preparation. Here, I present a method for stabilizing mitochondria using formaldehyde, a reagent commonly used for cell and tissue preservation. In this project I explore the effects of formaldehyde treatment on stabilizing the electrophoretic properties of isolated mitochondria. Isolated mitochondria from L6 cells are treated with 4% formaldehyde and the distributions of the electrophoretic mobility are measured using CE-LIF and compared to those obtained from untreated mitochondria samples. If successful, stable samples of mitochondria will allow long-term storage and transportation of mitochondria, which will lead to advancements in the fields of mitochondrial research and particle separation by CE-LIF.