Gibco™ MEM α, Nucleosides

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	supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, U.S.A.) and antibiotics (100 units/ml of penicillin and 100 µg/ml of streptomycin).

Protocol tips
supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, U.S.A.) and antibiotics (100 units/ml of penicillin and 100 µg/ml of streptomycin).

Publication protocol

Virus isolation of RVA was conducted on MA-104 cells (ATCC CRL-2378.1). MA-104 cells were grown in T25 flasks with α-Minimum Essential Medium (α-MEM, Gibco, Tulsa, OK, U.S.A.) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, U.S.A.) and antibiotics (100 units/ml of penicillin and 100 µg/ml of streptomycin). Confluent MA-104 cell monolayers were used for virus inoculation. The inoculum was pretreated with trypsin (type IX, Sigma, St. Louis, MO, U.S.A.) at a final concentration of 10 µg/ml at 37°C for 60 min. Growth medium was removed, and the monolayers of cells were washed with PBS twice before inoculation. Then 0.5 ml of treated inoculum and 0.5 ml maintenance medium was added. Maintenance medium was consisted of α-MEM supplemented with antibiotics (100 units/ml of penicillin and 100 µg/ml of streptomycin) and 0.5 µg/ml trypsin (type IX, Sigma). Following virus adsorption for 60 min at 37°C, inoculum was removed and the cell monolayers were washed with PBS once. Finally, 5 ml maintenance medium were added to each flask. Cultures were incubated at 37°C with 5% CO2, and examined daily for cytopathic effect (CPE). When CPE appeared in more than 80% of cells (−5 days after inoculation), the flasks were subjected to two freeze-thaw cycles. The cells and supernatants were harvested and stored at −70°C. These samples were used as seed stocks for the next passage. Serial four blind passages were performed if no CPE appeared within 5 days.

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