Tour-de-Force

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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
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007The JC-1 probe is a monomer in the cytosol emits green light. The membrane potential attracts the probesto the inside of the mitochondria, where the probes form aggregates, called J-aggregates, and emit redlight. The higher the membrane potential, the higher the ratio of aggregates in the mitochondria, so thehigher the amount of red light emitted as compared to green light. This way, the ratio of red-to-green JC-1fluorescence can be calculated and so a change of membrane potential can be observed. This ratio isonly dependent on changes in membrane potential and not dependent of size, density etc. (Foster et al.2006).As such, a high ratio means that there are a lot of aggregates (red) inside the mitochondria relative tomonomers (green) in the cytoplasm and this means that there is a high membrane potential.Appendix 1.4A: Measuring mitochondrial membrane potential with a JC-1 probe.The JC-1 Mitochondrial membrane potential detection kit 30001 from Biotium () will beused and the supplied protocol will be followed.This measurement will be done 11 times in the hTERT-RPE 1 (retinal epithelium) cells. For the fibroblastthe amount of measurements depends on the doubling time; half the amount of the doubling time in hours+ 1 is needed as to be able to take measurements every two hours, starting at t=0, throughout one entirecell cycle.The entire protocol will be done 5 times in five separate synchronized cultures.Materials:0.3 µg/ml JC-1 diluted in assay buffer (Biotium)Fixator (4% formaldehyde)(Invitrogen)Flow cytometer with a 15 mW, 488 nm argon excitation laser (Utrecht University)11 wells with medium as described in Appendix 0 for the hTERT-RPE 1 cells, a number(dependent on outcome of experiment described in Appendix 0.A) of wells with medium asdescribed in Appendix 0 for the CCD-1072 Sk cells.Appendix 2.1: ROS probesProbes:Probe Lex Lem Incubation time Expression plasmidPF-1 490 530 5-10 Not expressedHyPer, HyPer-M 488 500-520 Gets expressed pQE30MitoSOX 510 580 10 Not expressedTable B.1 Overview of probe characteristics. Both PF-1 and MitoSOX will be dissolved in DMSO.Appendix 2.2: PF-1Besides being a ROS, superoxide also shows super-nucleophilicity. This characteristic allows forsuperoxide detection by 3’,6’-bis(diphenylphosphinyl)-fluorescein (PF-1), a phosphinate, since superoxidecauses increased hydrolysis of phosphinate. Probes will be synthesized as described in Xu et al. (2007).Growing cells on a coverslip (WEHI):• First the coverslip will be soaked in 70% EtOH• Then the coverslip will be placed into a 10 cm petridish containing the appropriate medium for thecell line (as described in Appendix 1)• The coverslip will remain in the dish for 30 minutes to allow the EtOH to evaporate• Then the cell can be added to the coverslip and transfected if neededTo obtain fluorescence data from single cell, a Zeiss 510 confocal laser scanning microscope will be used,with a 1.4 numerical aperture objective, and 125 mW argon and 1 mW helium-neon lasers. Scanning willbe performed using 400 Hz line frequency, 512 x 512 format. Excitation of the probe at 490 nm., emissionat 530 nm. Image intensities will be quantified with Zeiss confocal software.SCI 332 Advanced Molecular Cell Biology Research Proposal 105
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Tour-de-Force

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