Ex cell 405 medium

All PS-targeting proteins used in this study were produced using the Expi293 expression system from Life Technologies following the manufacturer’s instructions. Briefly, cells were transfected with expression constructs for 6–7 days and Fc fusion proteins were purified from culture supernatants using protein G-Sepharose. Bound proteins were eluted using 50 mM diethylamine with 150 mM NaCl. The eluted protein was neutralized using 2M Tris pH 7.0 followed by dialysis against PBS. All proteins were concentrated and loaded onto a Hiload 16/60 Superdex 200 gel filtration column (GE Healthcare). The homodimeric, non-aggregated form of the protein was separated, concentrated and analyzed using a Superdex 200 15/30 gel filtration column (GE Healthcare).
Recombinant mouse FcRn was produced as previously described (24 (link)). Briefly, High Five cells grown at 27°C in EX-CELL 405 medium (Sigma, catalog # 14405) were infected at a density of 1 x 10⁶ cells/ml with recombinant baculovirus (mouse FcRn α-chain/mouse β2-microglobulin). Cells were cultured at 23–24°C for 72 hours. Mouse FcRn was purified from the supernatant using Ni²⁺-NTA agarose (Qiagen) followed by the use of a Hiload 16/60 Superdex 200 gel filtration column.

Human embryonic kidney (HEK) 293T cells were maintained at 37° degrees Celsius with 5% CO₂ in DMEM supplemented with 10% FBS, penicillin and streptomycin. Human 293F cells were maintained at 37° degrees Celsius with 5% CO₂ in FreeStyle 293 Expression Medium (ThermoFisher) supplemented with penicillin and streptomycin. MS40L-low feeder cells (Mus musculus) were expanded from frozen aliquots in Iscove’s Modified Dulbecco’s Medium (Invitrogen) containing 10% HyClone FBS (Thermo scientific), 2-mercaptoethanol (5.5 × 10⁻⁵ M), penicillin (100 units/ml), and streptomycin (100 μg/ml; all Invitrogen) at 37° degrees Celsius with 5% CO₂ (Luo et al., 2009 (link); Su et al., 2016 (link)). Madin-Darby canine kidney cells (MDCK) (Canis lupus familiaris) were maintained in DMEM at 37° degrees Celsius with 5% CO₂. High Five^™ Cells (BTI-TN-5B1-4) (Trichoplusia ni) were maintained at 28° degrees Celsius in either EX-CELL 405 medium (Sigma) supplemented with penicillin and streptomycin or Express-Five (Thermo Fisher) medium supplemented with L-glutamine (Gibco) at a final concentration of 16 mM, penicillin and streptomycin. Cell lines were not subject to authentication.

Human 293F cells were maintained at 37° degrees Celsius with 5% CO₂ in FreeStyle 293 Expression Medium (ThermoFisher) supplemented with penicillin and streptomycin. MS40L-low feeder cells (Mus musculus) were expanded from frozen aliquots in Iscove’s Modified Dulbecco’s Medium (Invitrogen) containing 10% HyClone FBS (Thermo scientific), 2-mercaptoethanol (5.5 × 10⁻⁵ M), penicillin (100 units/ml), and streptomycin (100 μg/ml; all Invitrogen) at 37° degrees Celsius with 5% CO₂ (Luo et al., 2009 (link); Su et al., 2016 (link)). Madin-Darby canine kidney cells (MDCK) (Canis lupus familiaris) were maintained in DMEM at 37° degrees Celsius with 5% CO₂. High Five™ Cells (BTI-TN-5B1–4) (Trichoplusia ni) were maintained at 28° degrees Celsius in EX-CELL 405 medium (Sigma) supplemented with penicillin and streptomycin. EL4 cells and EL4 cells transduced with influenza HAs were maintained at 37° C with 5% CO₂ in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated HyClone FBS (Fisher Scientific), HEPES buffer (10 mM), sodium pyruvate (1 mM), 1× MEM NEAA, 2-mercaptoethanol (5.5 × 10⁻⁵ M), penicillin (100 units/ml), and streptomycin (100 μg/ml; all Invitrogen). Cell lines were not subject to authentication.