Flow Cytometry Labeling of HIV-1 Envelope Trimers

Biotinylated BG505 SOSIP MD39 trimer and BG505 SOSIP.v5.2 N241/N289 trimers were generated as previously described (17 (link), 23 (link)). Biotinylated proteins were individually premixed with fluorochrome-conjugated streptavidin (Brilliant Violet 650 or Brilliant Violet 421, BioLegend) at room temperature for 20 min. BG505 SOSIP MD39-ferritin and BG505 SOSIP T33-31 nanoparticles were generated and directly conjugated to Alexa Fluor 647 (Thermo Fisher Scientific). Cells were incubated with indicated probes for 30 min at 4°C, and then surface antibodies were added and incubated for 30 min at 4°C. Cells were washed and then sorted on a BD FACSAria II. The sorting procedure is shown in fig. S6.