Frequently requested material

We maintain these cell lines in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 500 µg/ml G418, 1 µg/ml puromycin and 1 µg/ml tetracycline. Media containing tetracycline should be kept protected from light. We split the cells 1:15 on a weekly basis. For maintenance purposes, cells are always cultured in medium containing tetracycline. To derepress the cells, i.e., to induce HCV protein expression, we rinse them twice with PBS. This should be done with careful aspiration of all media and washes since as little as 0.01 µg/ml tetracycline will still markedly repress the activity of the tTA-dependent promoter. We suggest that you always split the cells into medium containing tetracycline and let them settle and become about 80-90% confluent before you derepress them. After tetracycline withdrawal, HCV proteins become detectable by immunoblot after 4-6 h and expression reaches a steady state after 24(-48) h. We freeze the cells in 50% of the above-mentioned medium, 40% FBS, and 10% DMSO at a density of 1-5 Mio cells/ml.