Invited Symposium: Iron Transport
Materials and Methods
Cell survival and proliferation were evaluated using the MTT proliferation assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium) method is based on the reduction of the tetrazolium salt to the blue formazan product by mitochondrial dehydrogenases of living cells. Cells were seeded aseptically into a 96-well microtitre plate at a density of 10 000 / 110無 in MEM containing FCS (10% v/v) and diferric transferrin (1.25然). This density yields an exponential growth of cells in the absence of chelators over a 48h time period. After incubation with the chelators (0-500然), MTT (5mg/mL) was added and the cells incubated for two hours. Finally a solution of SDS (10%) and isobutanol (50%) in 0.1M HCl was added and the plates reincubated for another 2h at 37蚓 and the dissolved formazan product was then read at 595nm on a microplate reader.
Fe Uptake into the cells in the presence or absence of chelators was measured over a 24h time interval. Cells in the exponential phase of growth were incubated in MEM (pH 7.4) containing BSA (10mg/mL), the Fe chelator (500然) and 59Fe-Tf- (1.25然) for up to 24 h. Parallel measurements were made on primary cultures of adult rat hepatocytes. After incubation, the cells were washed, Pronase (1mg/mL) added and the cells incubated for a further 30 minutes at 4蚓. The suspension was then centrifuged (10 000 x g) to separate the membrane bound (Pronase sensitive) and internalised (Pronase resistant) Fe 59 fractions which were then measured on a LKB Wallac universal gamma counter. Results shown are for HTC cells and are representative of all cell lines.
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|Kicic, A; Baker, E; (1998). The Effects of Iron Chelators Upon Cellular Proliferation and Iron Transport in Hepatoma Cells.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/templeton/kicic0473/index.html|
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