Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Appendix BAppendix 0: Cell culture and treatmentTwo different cell types will be used throughout the project. CCD-1072 Sk human fibroblasts (CRL-2088,ATCC) have a relatively short doubling time and are capable of at least 28 more doublings. The hTERT-RPE 1 retinal epithelial cells (CRL-4000, ATCC) are immortalized using human telomerase reversetranscriptase (hTERT). They have a doubling time of about 19 hours.The fibroblasts will be cultured in 90% Iscove’s modified Dulbecco’s medium (I-6529, Sigma), 10% FBS(Sigma), streptomycin and penicillin (Sigma). For quiescent cells, FBS is omitted.The retinal epithelial cells will be cultured in a 1:1 mixture of modified Dulbecco’s medium (I-6529, Sigma)and Ham’s F12 medium (N-8641, Sigma) supplemented with 0.01 mg/ml of hygromycin B (H3274, Sigma)and 10% FBS. For quiescent cells, FBS is omitted.All cells are grown at 37 °C with 5% CO 2 at a PH of 7.4. Subculturing will be carried out according to theprotocol provided by ATCC.Appendix 0.A: Measurement of human fibroblast doubling timeThe doubling time of the human fibroblasts will be measured with a normal light microscope, simply bymonitoring the time span between two consecutive cell divisions. This will be measured for 10 differentcells and the average will be taken to represent the doubling time of these human fibroblastsAppendix 1.1: Cell synchronizationNocodazole is used to halt cells in the prometaphase after which all cells arrested in mitosis will beseparated from the other cells with mitotic shake-off.1.1a: Nocodazole treatmentThis experiment will be done with cultures of both cell types.400ng/ml nocodazole diluted in DMSO will be added to the cells and incubated at 37°C for 12-16 hours.The cells will be washed and treated according to the protocol described by Jackman and O’Connor(2001). After the treatment the cells will be transferred to a plate that can be used for mitotic shake-off.Materials:• 400ng/ml Nocodazole in DMSO (SIGMA)1.1b: Mitotic shake-offThe plate with the nocodazole treated cells will gently be tapped against a hard object (i.e. a desk) for 1minute and the detached cells will be collected in a culture flask according to the protocol of Jackman andO’Connor (2001). The shaken-off mammary epithelial cells will be divided over wells. For the hTERT-RPE1cells, with a cell cycle length of 19 hours, 11 wells are needed, since measurement time intervals of twohours are used. For CCD-1072 Sk cells, this depends on the outcome of the measurements described inAppendix 1A. Half the amount of the doubling time in hours + 1 is needed as to be able to takemeasurements every two hours, starting at t=0, throughout one entire cell cycle.Appendix 1.2: Cell-phase determinationSynchronized cells (as described in appendix 1.1) of both cell lines will be grown in wells in media asdescribed above. With BrdU pulse-labeling and Histone H3 phosphorylation the timing of the different cellphases will be determined.Length of G1 = End of mitotic shake-off until the start of BrdU-incorporationLength of S = Start of BrdU-incorporation until the end of BrdU-incorporationLength of G2 = End of BrdU-incorporation until the start of Histone H3 phosphorylationLength of M = Start of Histone H3 phosphorylation until the end of Histone H3-phosphorylation.1.2a: BrdU pulse-labelingSCI 332 Advanced Molecular Cell Biology Research Proposal 102
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007At time points of two hours 1µg/ml BrdU will be added to one of the wells for 30 minutes. Then the cellswill be fixated with 4% paraformaldehyde and nucleases will be added to cut open the DNA. Then anti-BrdU antibodies [BrdU (MBDU-65A): sc-65723 (Santa Cruz biotechnology, Inc.)] will be added and thefluorescence will be measured. For this experiment a BrdU-kit from Roche Applied Sciences (ELISA) willbe used and the supplied protocol will be followed.The cell phase can be determined with BrdU until the end of S-phase. When no BrdU-incorporation ismeasured anymore, Histone H3 phosphorylation method will be used.Materials:• 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit III (Roche Applied Sciences)• 1µg/ml BrdU in PBS (Roche Applied Sciences)• 4% paraformaldehyde• Anit-BrdU antibodies [BrdU (MBDU-65A): sc-65723 (Santa Cruz biotechnology, Inc.)]• Nucleases (Roche Applied Sciences)• ELISA reader1.2b: Histone H3 phosphorylationThe onset of mitosis and length of G2 will be determined with Histone H3 phosphorylation. When cellsenter mitosis, histone H3 is phosphorylated at the Ser10 residue while at other times in the cell cycle itremains unphosphorylated.Cells will be fixed in 70% ethanol overnight at –20°C. Subsequently, they will be permeabilized with PBScontaining 0.1% Triton X-100 for 20 minutes at 4 °C, blocked with 2% FBS in PBS, and incubated with 1µg of anti-pSer10-histone H3 anti-rabbit antibody per 10 6 cells for 60 minutes on ice. After washing, cellswill be incubated with 1 µg of Alexa Fluor 488-conjugated goat anti-rabbit antibody per 10 6 cells for 30minutes on ice, washed and re-suspended in PBS.Cells will be prepared for flow cytometry following the protocol accompanying the anti-pSer10-histone H3antibodies. FACS will be carried out according to the protocol to count cells containing phosphorylatedhistone H3.Materials:• Ethanol (Sigma)• PBS containing 0.1% Triton X-100 (Invitrogen)• anti-pSer10-histone H3 anti-rabbit antibody (Cell Signaling Technology)• Alexa Fluor® 488 F(ab') 2 fragment of goat anti-rabbit IgG (Invitrogen)FACS for cell cycle phase checkPreparation of cells for FACS will be performed as described by Ezhevsky and colleagues (1997): Cellsarrested in prometaphase will be washed with PBS, fixed in 70% ethanol overnight at -20 °C, andrehydrated with PBS containing 0.1% BSA, RNAase A (1 µg/ml) and propidium iodide (10 µg/ml), 20minutes before analysis with FACS. For FACS procedure, the protocol accompanying the machine atUtrecht University will be followed.Appendix 1.3: Oxygen measurementThis method follows the protocol designed by BioCurrents Research Center (BRC) and described in Smithet al (2007) and Osbourn et al. (2005).A self-referencing microelectrode with a 2µm platinum diameter reactive surface at the tip will be usedalong with the return path reference electrode (3M KCl in 3% Agar). The amperometric electrode will bemade following the protocol of BioCurrents Research Center (MBL, Woods Hole MA: “Electrodeconstruction” 2007; Smith, Sanger and Messerli 2007; Messerli, Robinson and Smith 2006: &www.biocurrents.org). The self-referencing microelectrode will be constructed on an inverted microscope,fitted with DIC and/or phase contrast capability. The microscope is mounted on a vibration isolation tableand housed in a Faraday box. Images are acquired remotely via a video port. The appropriatemeasurement site will be determined visually and although cell specific is normally taken away from thenucleus where more mitochondria are located. An example of this, with the application of self-referencingis given by Osbourn et al (2005) The electrode will be attached to a 3 dimensional stepper motorSCI 332 Advanced Molecular Cell Biology Research Proposal 103
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