Freestyle 293 f suspension cells

Expression of His-tagged PD-1 wild-type protein and its alanine mutants was carried out using FreeStyle 293-F suspension cells (Invitrogen). Cells were grown in FreeStyle 293 Expression Medium (Invitrogen) and maintained at 37 °C, 80% humidity, and 8% CO₂ in an orbital shaker at 130 rpm. Eighty milliliters of cell culture was transiently transfected with 100 µg of PD-1 plasmid using Polyethylenimine Max Transfection Reagent (PolySciences) in 150 mM sodium chloride solution. The culture medium was then harvested for 7 days after transfection.

The recombinant gp120-BC protein was produced by transient transfection of FreeStyle™ 293F suspension cells (Invitrogen, Carlsbad, CA) [9 (link)] using the same gp120-encoding DNA vaccine plasmid. In brief, cells were transfected at a density of 1 × 10⁶/ml in GIBCO® FreeStyle™ 293 expression media using 293fectin™, according to manufacturer’s instructions (Invitrogen). Three days after the transfection, supernatant was collected and the gp120 protein was purified by lentil-lectin affinity chromatography (GE Healthcare, Chicago, IL). The purified gp120-BC protein was verified by ELISA and Western-blot analysis.

IgH and IgL DNA fragments coding for human MDE8 (41 (link)) antibody were prepared by PCR-amplification from codon-optimized synthetic genes (Life Technologies, Thermo Fisher Scientific). Purified digested DNA fragments were cloned into human Igγ1-and Igλ-expressing vectors (42 (link)), and human MDE8 IgG1 antibod-ies were produced by transient co-transfection of Freestyle™ 293-F suspension cells (Thermo Fisher Scientific) using PEI-precipitation method as previously described (43 (link)). Recombinant IgG1 antibodies were purified by batch/gravity-flow affinity chromatog-raphy using protein G sepharose 4 fast flow beads (GE Healthcare, Chicago, IL) according to the manufacturer’s instructions, extensively dialyzed against PBS using Slide-A-Lyzer^® dial-ysis cassettes (Thermo Fisher Scientific) and quantified using NanoDrop 2000 instrument (Thermo Fisher Scientific) (43 (link)).

Codon-optimized nucleotide fragments encoding a stabilized version of SARS-CoV-2 WA1 or Omicron BA.1 spike (HexaPro) ectodomain (followed by a foldon trimerization motif) and WA1 or Omicron BA.1 RBD proteins containing C-terminal tags (Hisx8-tag, Strep-tag, and AviTag) were synthesized and cloned into pcDNA3.1/Zeo⁽⁺⁾ expression vector (Thermo Fisher Scientific). Recombinant proteins were produced by transient transfection of exponentially growing Freestyle 293-F suspension cells (Thermo Fisher Scientific, Waltham, MA, USA) using polyethylenimine (PEI) precipitation method as previously described (PMID: 25910833). Proteins were purified from culture supernatants by high-performance chromatography using the Ni Sepharose^® Excel Resin according to manufacturer’s instructions (GE Healthcare, Chicago, IL, USA), dialyzed against PBS using Slide-A-Lyzer^® dialysis cassettes (Thermo Fisher Scientific), quantified using NanoDrop 2000 instrument (Thermo Fisher Scientific), and controlled for purity by SDS-PAGE using NuPAGE 3–8% Tris-acetate gels (Life Technologies, Carlsbad, CA, USA), as previously described (PMID: 25910833).

Mycoplasma-free spontaneously immortalized human keratinocyte (HaCaT) cells (83 (link)), HaCaT cells stably expressing pUL21 (HaCaT21) (18 ), African green monkey kidney (Vero) cells (American Type Culture Collection; catalog no.: CRL-1586), and HEK293T cells (American Type Culture Collection; catalog no.: CRL-3216) were maintained in Dulbecco’s modified Eagle's medium (DMEM) with high glucose (Merck), supplemented with 10% (v/v) heat-inactivated fetal calf serum and 2 mM l-glutamine (complete DMEM) in a humidified 5% CO₂ atmosphere at 37 °C. For protein purification, Freestyle 293F suspension cells (Thermo Fisher Scientific) were grown in Freestyle 293F medium (Gibco) on a shaking platform (125 rpm) in a humidified 8% CO₂ atmosphere at 37 °C.
Doxycycline-inducible stably transfected Freestyle 293F cells expressing StrepII-CERT_L and StrepII-CERT_L^S132A were generated using a piggyBac transposon-based system (80 (link)). A 30 ml suspension culture of Freestyle 293F cells at 1 × 10⁶ cells/ml was transfected with a 5:1:1 mass ratio of PB-T-CERT_L^(S132A):PB-RN:PBase (35 μg total DNA) using Freestyle MAX transfection reagent (Invitrogen) as per the manufacturer’s instructions. After 2 days, the cells were transferred to fresh media supplemented with 500 μg/ml geneticin (Gibco), and the drug selection was continued for 2 weeks with media replenishment every 3 days.