Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 20072. Incubation with MitoSOXThe cell cultures will be incubated with BrdU and MitoSOX dissolved in DMSO at two-hour time intervals,starting at t=0. As a control, one cell culture will only receive DMSO treatment.3. Fixation & Measuring fluorescence and cell cycle progressionAfter the appropriate incubation time for BrdU and MitoSOX, the cells will be washed with PBS and fixatedby incubation with formaldehyde. Afterwards these cells will be examined under a confocal laser scanningmicroscope to determine the level of fluorescence emitted by MitoSOX. Again this will be performed onfive different single cells. The level of BrdU-incorporation of the cells will also be determined to establishtheir cell cycle progression, as in experiment 2.1.2. Intensity will then be semi-quantified using theappropriate software.Experiment 2.1.4: Mitochondrial H 2 O 2 levels throughout the cell cycleThe HyPer probe can be targeted to different organelles using the different targeting sequences.Therefore, in this experiment to measure mitochondrial ROS levels, the probe called HyPer-M, which istargeted to the mitochondria, will be used. This probe is a normal HyPer probe using the mitochondriallocalization sequence. (See protocol of Belousov et al., 2006).1. Construction of HyPer-M expression vectorA HyPer expression factor will be constructed and checked according to protocol as described byBelousov et al. (2006) explained in Appendix B2.3.2. Plating and growing cells on a coverslipThe cells will be grown on a coverslip as indicated in Appendix B2.2.3. Cell cycle controlAs a control, thirteen cell cultures will be taken from the same synchronized batch as the cells which areplated on a coverslip. These cells will then be plated in wells with appropriate medium and every twohours a different well will be checked for its cell cycle progression by BrdU.4. Transfecting the cell with HyPer-MThe cells will be transfected with HyPer-Mito expression vector, as explained Appendix B2.3. To controlfor the effects of transfection on the cells, a different cell culture will be mock-transfected.5. Measuring fluorescenceThe same procedure as in experiment 2.1.2 will be used.Experiment 2.1.5: Peroxisomal H 2 O 2 levels throughout the cell cyclePeroxisomes mainly produce hydrogen peroxide (Schrader and Fahimi, 2006). This experiment thereforemeasures the levels of H 2 O 2 produced by peroxisomes throughout the cell cycle.1. Construction of peroxisomal HyPer expression vectorA HyPer expression factor will be constructed and checked according to protocol as described byBelousov et al. (2006) explained in Appendix B2.3.2. Plating and growing cells on a coverslipThe cells will be grown on a coverslip as indicated in Appendix B2.2.3. Cell cycle controlAs a control thirteen cell cultures will be taken from the same synchronized batch as the cells which areplated on a coverslip. The procedure is the same as that of experiment 2.1.2 and 2.1.3.4. Transfecting the cell with peroxisomal HyPerThe cells will be transfected with HyPer-Peroxi expression vector, as explained in Appendix B2.3. Tocontrol for the effects of transfection on the cells, a different cell culture will be mock-transfected.SCI 332 Advanced Molecular Cell Biology Research Proposal 20