Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Cell FractionationCell fractionation refers to a broad set of techniques aiming to separate and isolate specific organellesfrom cell. In other words, it is the separation of homogenous sets (organelles) from a heterogenouspopulation of cells. There are a number of steps involved in cell fractionation; namely disruption (alsoknown as homogenization) of cells, macro-filtration and purification of the cell components.Central-ElutriationCentrifugal elutriation is a technique used to synchronize cells at specific phases of the cell cycle. It is adrug-independent method capable of synchronizing cells from asynchronous cell populations in any phaseof the cell cycle. This method entails that cells in a particular phase in the cell cycle can be isolatedaccording to their size. In other words, early G1 cells are around half the size of cells in late G2 or Mphase. However, it is important to consider that not all cell types show significantly enough size variabilitythroughout the cell cycle.Cerium Ions with ReflectanceConfocal laser scanning microscopy adaptations have been made to allow detection and assessment ofsemiquantitatively cerium based primary reaction products of oxidases, phosphatases as well as the 3,3-diaminobenzidine (DAB) based primary reaction product of cytochrome-c-oxidase. This technique offers aunique method to make histochemical reaction products, which are weakly absorbent, but sufficientlyreflective, visible.ChemiluminescenceChemiluminescence is the generation of light by a chemical reaction. When used as a method, a specificprobe can be used, which reacts with a specific compound. The luminescence can then be measured by aluminometer, to show and quantify the presence of the compoundCo-immunoprecipationCo-immunoprecipation can be used to check if proteins physically interact with each other and form animmune complex. First, the cell gets lysed, after which antibodies get added, which specifically bind to theprotein complex. Then the antibodies and the bound immune complex get immobilized on a solid complex,after which the remaining cell lysate is washed off. The complexes are then eluded and analyzed by SDS-PAGE usually followed by a western blot.Figure A.1: Principles of co-immunoprecipitationSCI 332 Advanced Molecular Cell Biology Research Proposal 96
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Compound C Specific InhibitorA specific inhibitor called compound C inhibits AMPK and is produced by Merck UK. This can be usedaccording to the manufacturer’s protocol.Confocal Laser Scanning MicroscopyConfocal laser scanning microscopy is a technique for obtaining high-resolution optical images and can beused to semi-quantify the fluorescence. A laser beam passes through a light source aperture and then isfocused by an objective lens into a small focal volume within a fluorescent specimen. The specimen thenemits reflected laser light and emitted fluorescence, then the laser light gets filtered out so that thefluorescence can be absorbed by a detection apparatus.Double Thymidine BlockIs used for cell synchronization, by adding excessive amount of thymidine, so that DNA replication isblocked. That way all cells will be halted in G1/S phase, and this arrest is lifted by adding additionalguanine, adenine and cytosine.Extracellular Flux (FX)FX is a method to measure the changes in oxidative phosphorilation and glycolysis. The measurementsare done by observing alterations in the rates of consumption or production of extracellular solutes relatedto aerobic and anaerobic cellular metabolism, such as oxygen, protons, nutrients, carbon dioxide, lactate,or lactic acid. The measurements can be done by means of a XF24 Extra Cellular Flux Analyzer.Fluorescence Activated Cell Sorting (FACS) AnalysisCells in a solution are led through a small nozzle one by one in droplets, passing a fluorescencemeasuring apparatus. This measures the fluorescence of proteins contained in the cell. A computeranalyzes the data and separates droplets with different fluorescent properties by applying a small currentto the droplets, which are then deflected into tubes by passing electrodes. The data measured can beused by computer analysis to give quantities of cells with the different fluorescent properties.Glucose DeprivationDenying the cell glucose in order to prevent glycolysis. To do this, a glucose-free Dulbecco's modifiedEagle's medium can be bought and applied according to the manufacturer’s protocol from invitrogen.Growth Factor DeprivationDenying the cell growth factors in order to prevent cell cycle progression and cause quiescence. Toachieve this a growth-factor-free medium can be used.GST Expression and PurificationIn this technique, vectors are introduced into the cell so that they can stimulate high expression ofglutathione S-transferase (GST). The protein that needs to be purified is linked to GST with certaincleavage site so that they can be separated. Glutathione-affinity chromatography can be used to purifyfusion proteins from cell lysates.Immunofluorescence MicroscopyA certain protein of interest is targeted by an antibody, which contains a fluorescent chemical group.Following the labeling of proteins with fluorescent dyes, immunofluoresence microscopy is used tovisualize, localize and sometimes quantify protein of interest within the cell. Cells can be incubated withmore than one tag with different colors. Because cells are not killed as in electron microscopy, thistechnique can also be used in real-time measurements.Inhibition of ATP synthesis; Olygomycin & CCCPOxidative phosphorylation is one of the main processes to take place in mitochondria. Inhibition of thisfunction is used to understand the energy metabolism of the cell or to further study the oxidativephosphorylation event. Olygomycin decreases oxygen consumption to background values by inhibiting theenzymatic activity of ATP syntheses. CCCP inhibits oxidative phosphorylation by uncoupling the electronSCI 332 Advanced Molecular Cell Biology Research Proposal 97
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