Express five media

Schneider’s Drosophila line 2 (S2) cells, purchased from ATCC in the course of these experiments ([D. Mel. (2), SL2] (ATCC CRL1963); RRID:CVCL_Z232), were grown at 28°C in Schneider’s medium and 8% fetal bovine serum or in serum-free Express-Five media, supplemented with penicillin, streptomycin, and L-glutamine (Gibco). Passages 2–4 of this ATCC stock were frozen and on a regular basis, portions of these early passages were thawed to seed new batches of cells, yielding results that were highly reproducible. Sucrose gradient-purified FHV stocks were produced as previously described (Friesen and Rueckert, 1984 (link)). Drosophila S2 cells were infected at a multiplicity of infection of 10 for all experiments and allowed to proceed for 16 hr unless otherwise noted. DNA plasmid transfections of S2 cells used kit V and G-030 pulse settings on an Amaxa nucleofector (Lonza) or two 30 ms pulses of 1200 V in 100 µL tips on a Neon electroporator (Bio-Rad) and were harvested 48–72 hr post-electroporation.

High Five cells (BTI-TN-5B1–4, Trichoplusia ni) were grown in serum-free Express Five media (Gibco) containing 10% L-Glutamine and 1% penicillin/streptomycin antibiotics mix. Sf9 cells (Spodoptera frugiperda) adapted from the cell line ATCC CRL-1711 were maintained in Trichoplusia ni medium—Fred Hink (TNM-FH, Gemini Bioproducts) supplemented with 1% penicillin/streptomycin antibiotics mix, 1% pluronic F-68 (Sigma-Aldrich), and 10% fetal bovine serum (FBS). In order to passage the baculoviruses, the media was switched to 3% TNM-FH (1% penicillin/streptomycin, 1% pluronic F-68, 3% FBS).

MDCK cells were cultivated in Dulbecco’s Modified Eagle’s Medium (complete DMEM, Gibco) containing 1% penicillin/streptomycin (100 U/ml of penicillin, 100 µg/ml streptomycin, Gibco) antibiotic solution, 10% fetal bovine serum (FBS, Gibco) and 1% hydroxyethylpiperazine ethane sulfonic acid (HEPES). SP2/0-Ag14 myeloma cells were propagated in complete DMEM supplemented with 1% L-glutamine (Gibco).
High Five cells (BTI-TN-5B1-4, Trichoplusia ni) were grown in serum-free Express Five media (Gibco) containing 1% L-glutamine and 1% penicillin/streptomycin antibiotics mix. Sf9 cells (Spodoptera frugiperda), adapted from the cell line ATCC CRL-1711, were maintained in Trichoplusia ni medium – Fred Hink (TNM-FH, Gemini Bioproducts) supplemented with 1% penicillin/streptomycin antibiotics mix, 1% pluronic F-68 (Sigma-Aldrich) and 10% fetal bovine serum. In order to passage the baculoviruses, the media was switched to 3% TNM-FH (1% penicillin/streptomycin, 1% pluronic F-68, 3% FBS).