Expi293 cells

All antibodies were expressed in Expi293 cells (Invitrogen) using standard manufacturer’s protocol. Expression cultures were typically grown for 5 days at 37 °C in 8% CO₂. Supernatants were harvested via centrifugation and sterile filtered. Proteins were affinity purified utilizing MabSelect Sure LX resin (GE Healthcare). For SPR screening, the IgV domains of human SIRPα v1 (NP_542970.1) and human SIRPα v2 (CAA71403.1) are expressed in Expi293 cells as described above as either a His-tagged or His-Avi-tagged fusions and purified using Ni-Sepharose 6 Fast Flow affinity purification and polished via gel filtration through a superdex hi-prep resin (GE Healthcare). The anti-SIRPα v1 specific antibody clone HEF-LB was generated as described in reference [23 ]. For crystallography, the IgV domain of SIRPα v1 and Fab fragments was generated as described [14 (link)].

Plasmids encoding prefusion RSV F (DS-Cav1), L141W, and D489Y variants and post-fusion (F ΔFP) proteins based on strain A2 were transfected into FreeStyle 293 F and Expi293 cells, respectively (Invitrogen, Carlsbad, CA, USA)^{32 (link), 34 (link)}. Proteins were expressed in the presence of kifunensine (5 μM) and were purified over Strep-Tactin resin (IBA, Goettingen, Germany). Prefusion RSV F (PRDM) protein^{36 (link)} for DSF studies was purified from transiently transfected Expi293 cells using a two-step purification protocol including cation-exchange chromatography at pH 5.0 (HiTrap Capto SP ImpRes column; GE Healthcare Biosciences, Pittsburgh, PA, USA) and size-exclusion chromatography using a Superdex 200 column (GE Healthcare). For crystallization studies, purification tags were removed by overnight digestion with thrombin followed by gel filtration using a Superose 6 column (GE Healthcare Biosciences) with a running buffer of 2 mM TRIS pH 8.0, 200 mM NaCl. For ITC studies, tags were not removed and proteins were purified by gel filtration using a Superdex 200 column (GE Healthcare) with a running buffer of PBS.

The heavy chain and light chain genes of FasMab and the rabbit anti-AFP mAb were subcloned into the pcDNA3.4 vector (Thermo Fisher Scientific). These mAbs were expressed in Expi293 cells (Thermo Fisher Scientific) according to the manufacturer’s instructions, and purified from clarified and filtered Expi293 cells culture supernatant using MabSelect SuRe (GE Healthcare). The in-house developed mouse anti-AFP mAb Clone 2B12 was purified from conditioned media of the hybridoma. Concentrations of all three mAbs were determined by measuring absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific).