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The stable replicon-containing cells were selected and maintained in medium containing 1 mg/mL G418 geneticin (Invitrogen, USA).
They were maintained in medium supplemented with E2.
For cell proliferation assays, transfected cells were maintained in medium without antibiotic selection.
Controls were performed with cells maintained in medium with 5% heat inactivated FCS for 6 h.
Single cell clones were maintained in medium with 10 µg/ml puromycin.
Cells were washed and maintained in medium free of growth factors for 7 days.
To avoid prestimulation by serum withdrawal, primary fibroblasts were maintained in medium with 0.5% FCS prior to stimulation with TNF.
The cells were then maintained in medium containing 100 µg mL−1 hygromycin and 15 µg mL−1 blasticidin.
Chemically selected stable cell lines were maintained in medium containing 0.5 mg/ml Geneticin or stored in liquid nitrogen.
The resistant pools were maintained in medium without Tzb for at least 2 days before each experiment.
Prior to their use in suppression assays, RC were thawed and maintained in medium plus 50 IU/mL of IL-2 for 48 h.
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