(526f) Developing a Method for the Isolation of Intact Viable Giant Mitochondria Utilizing An Electric Field

Scott P. Rose, Vratislav Kostal and Edgar A. Arriaga (Department of Chemistry, University of Minnesota-Twin Cities)

Introduction and Background:  Observable changes in mitochondrial morphology occur in shape and size as the organism ages.  Giant mitochondria can range between 1-5 µm, but the ability to analyze this unique organelle has been limited due to their fragile nature.  Current methods of organelle isolation deal with mechanical homogenization, which creates a powerful shear force that work well to release sub-cellular components, but tear apart the giant mitochondria and subsequently make analysis not possible.  We propose that the use of an electric field, applied with an electroporator, in conjunction with a solution containing digitonin, trypsin and colchicine will allow us to safely remove the plasma membrane and cytoskeleton from the mitochondria, thereby keeping the organelles of interest viable and available for study.

                Method:  10-N-nonyl acridine orange NAO labeled L6 rat myoblasts are being studied in a mitochondrial-isolation buffer solution in combination with trypsin, colchicine and digitonin.  Sample solution is then exposed to 90, 0.35kV pulses from an electroporator.   After electroporation, cells are then observed using fluorescent microscopy and flow cytometry or Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF) to determine cell count and number of intact mitochondria.

                Preliminary Work:  Our current method, using 90 short, electrical chain pulses disrupts upwards of 90% of the plasma membranes. The presence of mitochondria in an isolated subcellular fraction can be verified by flow cytometry and Capillary Electrophoresis with Laser Induced Fluorescence. Further analysis is being conducted using Flow Cytometry to identify the presence of giant mitochondria separate from whole cells and regular sized mitochondria.