Tour-de-Force

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