HIV-1 Env Gene Transfection and Antibody Staining

293 T cells were transfected with 1 ug of plasmid DNA encoding an HIV-1 env gene (PolyJet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories, Cat. No. SL100688) in 6-well plates and harvested at 48 h. Cells were split into separate staining reactions for monoclonal antibodies 3BNC117 (Cat. No. ARP-12474), 10-1074 (Cat. No. ARP-12477), 10E8 (Cat. No. ARP-12294), 447-52D (Cat. No. ARP-4030, all from BEI resources), b12 (Polymun, Cat. No. AB011), PGDM1400^{15 (link)} and PGT151^{93 (link)} (both kindly provided by Dr. Devon Sok and International AIDS Vaccine Initiative). 2G12 was produced in Expi293F cells from publicly available heavy and light chain sequences^{94 (link)} and purified using Protein A affinity chromatography. Cells were washed and stained with anti-human IgG-PE at 1:250 dilution (SouthernBiotech, Cat. No. 2040-09) for 15 min and fixed in 0.5% paraformaldehyde. Fluorescence was measured on a FACSymphony A5 SE Cell Analyzer and analyzed in FlowJo v.9.9.6.