Sf 900 serum free medium

Human embryo kidney (HEK) 293 T (ATCC CRL-1573) and Vero 76 (ATCC CRL-1587) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 50 μg/mL gentamycin and 10 % fetal bovine serum. PichiaPink Strain 1 (Invitrogen) was grown in YPD medium. Insect Sf9 cells (Gibco) were grown in Sf-900 serum-free medium (ThermoFisher Scientific).
PEDV strain USA/Colorado/2013 (GeneBank accession no. KF272920) was provided by Diagnostic Virology Laboratory (NVLS, Ames, USA), and the virus was propagated on Vero 76 cells in DMEM containing 50 μg/mL gentamycin, 2 μg/mL TPCK-trypsin and 0.2 % bovine serum albumin.

The Sf9 cells were cultured overnight, and the cell suspension was prepared using SF-900 serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA). Monolayer cultures consisting of 10⁶ cells were prepared in petri dishes with a bottom area of 35 mm. The successfully constructed transient expression plasmids were mixed with Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA) and transfected into Sf9 cells as required.