Gibco freestyle 293 expression medium

rIL4-10 FP was produced by transient transfection of HEK293F cells with a
pcDNA3.1-neo expression vector (Invitrogen, Carlsbad, CA, USA) containing a
dual CMV (cytomegalovirus) promoter. The vector contained 2 transgenes: cDNA
coding for rIL4-10 and cDNA coding β-galactosidealpha-2,3-sialyltransferase
to optimize glycan capping with sialic acid. To enable purification, a
hexa-histidine affinity tag was cloned on the N-terminus of rIL4-10 FP.
Cells were cultured in GIBCO FreeStyle 293 Expression Medium (Invitrogen).
The medium contained no serum or antibiotics. Cells were grown in flasks on
a shaker platform in humidified, 5% CO₂ cell culture incubator at
37 °C. Cells were split 3 to 4 times prior to transfection and they were
transfected at the cell viability of 90% and 1 million cells/mL cell
density. The transfection reagent used was 293fectin (Invitrogen). Culture
supernatant was harvested 72 hours after transfection.

Soluble recombinant proteins were produced by transient transfection of HEK293-6E mammalian cells grown in Gibco FreeStyle 293 expression medium (catalog no. 12338026, Thermo Fisher Scientific) as previously described (41 (link),42 (link)). Prey proteins were produced by transfecting 0.5 μg of DNA per million cells, and the conditioned medium was harvested 5 days after transfection. Prey protein expression levels were normalized using their β-lactamase activity to levels required for the AVEXIS assay, essentially as described (41 (link)). For the expression of biotinylated bait proteins, the bait plasmid was cotransfected with a plasmid encoding a secreted version of the biotin-ligase (BirA) (43 (link)) and dialyzed overnight in phosphate-buffered saline (PBS) to remove the unconjugated d-biotin in the medium. Baits were normalized by a standard ELISA using a monoclonal antibody (OX68) recognizing the Cd4d3+4 tag (32 (link)).

Human osteosarcoma U2OS and embryonic kidney fibroblast 293T cells obtained from ATCC were cultured in modified McCoy’s 5A medium (Thermo Fisher) and high-glucose Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% GlutaMAX (Thermo Fisher), and 1% penicillin-streptomycin (Pen-Strep; Thermo Fisher), respectively. The grivet kidney Vero cells (from ATCC) were cultured in DMEM supplemented with 2% FBS, 1% GlutaMAX, and 1% Pen-Strep. Human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured in EGM medium supplemented with EGM-SingleQuots (Lonza). Huh-7.5.1 cells were cultured as described above for 293T cells, with the addition of 1% nonessential amino acids. All adherent cell lines were maintained in a humidified 37˚C, 5% CO₂ incubator. Freestyle-293-F suspension-adapted HEK-293 cells (Thermo Fisher) were maintained in GIBCO FreeStyle 293 expression medium (Thermo Fisher) using shaker flasks at 115 rpm, 37˚C, and 8% CO₂.

Suspension-adapted FreeStyle™ human embryonic kidney HEK293-F cells (American Type Culture Collection Manassas, VA, USA) were routinely cultured in GIBCO FreeStyle™ 293 expression medium (Thermo Fisher Scientific) at densities of 0.1 × 10⁶ to 3.0 × 10⁶ cells/ml )more than 90% of the cells were viable) in disposable Erlenmeyer tissue culture flasks with vented caps (TriForest, Irvine, CA, USA) at 125–135 rpm on an orbital shaker incubator (37 °C, 8% CO₂). Cell density and viability were determined using a Countess II FL Cell Viability Analyzer (Thermo Fisher Scientific) according to the manufacturer's protocols.
HEK 293-T cells [American Type Culture Collection (ATCC), Manassas, VA, USA] and head and neck SCC47^{38 (link)} cells (kindly supplied by Reidar Grénman, Department of Otorhinolaryngology—Head & Neck Surgery, Turku University, Finland) were grown in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Haemek, Israel). Glioblastoma U87MG cells (ATCC) were grown in MEM medium (Biological Industries). These two media were supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin (Biological Industries), referred to below as "plus supplements". The cells were placed in an incubator at 37 °C and 5% CO₂.

All plasmids and primers used in this study are summarized in Supplementary Tables 1 and 2, respectively. All restriction endonucleases, T4 DNA ligase, and Vent polymerase were purchased from New England Biolabs (Ipswich, MA, USA). The Taq polymerase and oligonucleotide primers were from Biosesang (Seongnam, Republic of Korea) and Cosmogenetech (Seoul, Republic of Korea), respectively. Difco^TM Terrific Broth, Ni-NTA agarose, Protein A agarose, and glutathione agarose 4B were obtained from Becton Dickinson Diagnostic Systems (Sparks, MD, USA), Qiagen (Hilden, Germany), Genscript (Scotch Plains, NJ, USA), and Incospharm (Daejeon, Republic of Korea), respectively. An Alexa Fluor 488 labeling kit, 1-Step Ultra-TMB substrate solution, GIBCO FreeStyle™ 293 expression medium, and sheep anti-hC1q-HRP conjugate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyethyleneimine-Max and goat anti-GST-HRP conjugate were obtained from Polysciences (Taipei, Taiwan) and GE Healthcare (Piscataway, NJ, USA), respectively. Unless stated otherwise, all other biochemical reagents were purchased from Sigma‒Aldrich (St. Louis, MO, USA).