Author: Stewart, Meredith E.; Roy, Polly
Title: Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme Document date: 2015_2_24
ID: vzel6r43_4
Snippet: BSR cells (BHK-21 sub-clone) were maintained in Dulbecco modified Eagle medium (DMEM, Sigma Aldrich) supplemented with 5% (v/v) fetal calf serum (FCS, Invitrogen). The stable BSR-VP4 (BSR expressing VP4; BSR4) cell line was grown in DMEM-5% FCS supplemented with 7.5 lg/ml of puromycin (Sigma Aldrich). Cells and viruses were grown at 35°C in 5% CO 2 incubator. BTV-1 wild-type (WT) and mutant virus stocks were propagated by infecting BSR cells at .....
Document: BSR cells (BHK-21 sub-clone) were maintained in Dulbecco modified Eagle medium (DMEM, Sigma Aldrich) supplemented with 5% (v/v) fetal calf serum (FCS, Invitrogen). The stable BSR-VP4 (BSR expressing VP4; BSR4) cell line was grown in DMEM-5% FCS supplemented with 7.5 lg/ml of puromycin (Sigma Aldrich). Cells and viruses were grown at 35°C in 5% CO 2 incubator. BTV-1 wild-type (WT) and mutant virus stocks were propagated by infecting BSR cells at MOI of 0.5 and harvested when cytopathic effect (CPE) between 95% and 100% was evident. Titres of viral stocks were obtained by either plaque assay and/or by TCID 50 /ml. Spodoptera frugiperda (Sf9) cells were grown in either Sf900 III (Gibco BRL) or InsectExpress (Lonza) media supplemented with 2% (v/v) fetal calf serum and incubated either in suspension or in monolayer cultures at 28°C. Recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) that expressed either recombinant BTV-10 VP4 or mutated VP4 was propagated in Sf9 suspension culture.
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