Plasma Membrane Potential Estimation By Way of a Fluorescent Plate Reader : a Troubleshoot
Ralstrom, Christopher C.
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Drug uptake is an important aspect of many chemotherapeutic treatments, and more specifically in treatments that target the mitochondria. Drug uptake is directly affected and controlled by the plasma membrane, which creates an electrochemical difference that is referred to as plasma membrane potential. As a result of the fact that plasma membrane potential controls drug uptake, as well as the uptake of most substances exiting and entering the cell, the manipulation of the plasma membrane potential can lead to a manipulation in drug uptake. To manipulate drug uptake, it is necessary to first know what the plasma membrane potential is. One of the most common methods by which plasma membrane potential is measured is flow cytometry. Although effective, flow cytometers are extremely expensive to obtain and maintain. In 2010, Dr. Rod Braun adapted protocols used in flow cytometry for use on a fluorescent plate reader. This was achieved by comparing the fluorescent intensity of fixed and unfixed cells incubated in the fluorescent dye DiBAC4(3). The goal of this study was to optimize the methods adapted by Dr. Braun. During the optimization two problems arose; the lack of a difference in the fluorescent intensity between cells and the medium, and the similar uptake of DiBAC4(3) in fixed and unfixed R3230Ac cells. To address these problems and determine their cause, variations were made to the cell concentrations, fixatives, fixation times, and incubation media. These variables were ruled out as causative factors, however two other unexplored variables, e.g., a faulty plate reader or non-anionic DiBAC4(3), leave the possibility that this method may still be able to be optimized.