Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007assembly controlled remotely by a computer through Ionview software. The electrode tip is placedapproximately 5um from the visible edge of the cell or over the cell after briefly and gently touching thesurface before a measured withdrawal. This position is termed the near pole. The software will thencontrol the regular translation in a step function from the near pole to the far pole routinely set at 10umaway. In each position the current generated from the reduction of oxygen at –0.6V is sampled at 1000times per second, binned, averaged and compared to the values at the previous pole (Ionview). One thirdof all data is discarded to avoid artifacts during and after the movement to a new pole. Most commonly thespace and positioning of the electrode is maintained by visual observation or occasional checks by gentlytouching the cell. These are tedious but adequate approaches. Osbourn et al (2005) have published apreliminary report on a method for automating this process.The reference electrode is placed in the bulk medium.By moving the electrode between two points that are 10µm apart, at a frequency of 0.3 Hz, the sensor canmeasure the flux of oxygen into the membrane. The movement is controlled by the computer driven motorcontrol system as described in Figure B.1. The oxygen flux is calculated with the Fick equation: J= –D(∆C/ ∆X) where J = flux, D is the diffusion coefficient, ∆C is the differential concentration and ∆X is thedistance between the two electrode measuring positions (Smith et al 2007; Porterfield, 2007).From the well, one single cell will be selected that is as isolated as possible (it should have no contact withother cells). The oxygen consumption will be measured for 5 minutes every hour for one single cell,throughout the duration of one entire cell cycle.First of all, the measurements will be done 5 times in both cell lines. Secondly, the measurements will bedone in non-synchronized cells of both cell lines, as a nocodazole control. Thirdly, the measurements willbe done in non-proliferating cells, as a cell proliferation control.Materials:• BRC can provide a complete self-referencing system – stepper motors, motion controller,headstage preamplifier, main amplifier, A to D board, software and computer• Amperometric electrodes (BRC)• Return-path reference electrodeFigure B.1 Left: the electrode is place 5µm from the cell membrane and moves to and back from a point at 15 µmfrom the membrane. A reference electrode is placed in the bulk medium. In this figure the analyte can move throughchannel openings. Right: the electrode is attached to a motor and connected to a computer that monitors the data andcontrols the motor. In this figure analyte is injected into the medium. Adapted from BioCurrents Research Center©Appendix 1.4: JC-1 Dual-emission potential-sensitive probe to measure mitochondrialmembrane potential in single cellsSCI 332 Advanced Molecular Cell Biology Research Proposal 104