Vented erlenmeyer shake flasks

HEK293 cells (293-F, Invitrogen) were cultivated and transfected in suspension in serum-free FreeStyle 293 medium (Gibco). Cells were maintained and expanded in vented Erlenmeyer shake flasks (Corning) at 37°C and 8% CO₂ in a shaking incubator (Kuhner) set to 125 RPM and a 2″ shaking diameter. A mammalian expression vector was constructed by restriction sub-cloning the EcoRI/XbaI fragment used to produce pFastBac-CHIKV37997 into a pV1JNS-based [35] (link) plasmid under control of the hCMV promoter to create pV1JNS-CHIKV37997. This expression vector was transfected into HEK293 cells using 293fectin (Invitrogen) and the manufacturer-supplied protocol to produce positive control cells and culture supernatants containing CHIKV structural proteins and VLPs, respectively. Mock transfections with the pV1JNS vector (CHIKV37997 cassette omitted) were utilized as negative controls for immunofluorescence and protein analysis methods. Cell counts and cell diameters were determined using a Vi-CELL XR and accompanying image analysis software (Beckman Coulter) using the pre-loaded HEK293 image analysis algorithm.

Serum-free Sf-900II medium (Gibco) was obtained at a pH of 6.3 and was adjusted to different target pH levels: 1 N HCl (Sigma-Aldrich) was used to reduce pH to 6.0, and 1 N NaOH (Sigma-Aldrich) was used to increase pH to 6.6–6.8. Growth medium pH was measured using a calibrated pH meter and probe (Fisher Scientific Accumet), and the pH-adjusted medium was sterile filtered through a 0.2 μm Durapore membrane (EMD Millipore). Sf21 cells were centrifuged at 200× g, spent Sf-900II media was fully aspirated, and the cells were re-suspended in pH 6.0–6.8 formulations of Sf-900II. Re-suspended Sf21 cultures (at 3×10⁶ viable cells/mL) were inoculated with AcMNPV-CHIKV37997 in Sf-900II media at an MOI of 1 pfu per viable cell. 150 mL cultures were inoculated in 500-mL vented Erlenmeyer shake flasks (Corning). Inoculated cultures were incubated at 27°C in a shaking incubator (Kuhner) set to 80 RPM and a 2″ shaking diameter. Cell suspension samples were removed 72 hours post-infection for immunofluorescence flow cytometry. Harvest samples were removed 96 hours post-infection, centrifuged to remove cells, and submitted to qELISA analysis. Statistical analysis was performed using Minitab 16 software (Minitab).