Live dead fixable violet dead cell dye

Biotinylated BG505.T332N.SOSIP and DU156.12.SOSIP trimers were tetramerized via streptavidin-linked PE and APC (Invitrogen), respectively, as previously described (5 (link), 35 (link)). Peptide tetramer quality after conjugation was assessed by flow cytometry to a panel of well-characterized HIV-1–specific bnAbs. PBMCs from donor CH765 were stained with LIVE/DEAD fixable violet dead cell dye (Invitrogen), anti-human IgG (FITC), anti-human CD19 (Cy7-PE), anti-human CD3 (Cy7-APC), anti-human CD8 (BV711), anti-human CD14 (BV605), BG505.T332N.SOSIP trimer (PE), and DU156.12.SOSIP trimer (APC). PBMCs that were violet dye⁻, CD3⁻, CD8⁻, CD14⁻, CD19⁺, IgG⁺, and BG505.T332N.SOSIP⁺ and/or DU156.12.SOSIP⁺ were single cell–sorted using a BD FACSAria II into 96-well plates containing 20 μl of RT buffer, and cDNA synthesis was performed, as previously described (20 (link)). Ig heavy (V_H) and light (V_L) chains were PCR-amplified using a nested approach. First- and second-round amplification of V_H, V_κ, and V_λ genes was carried out as in (20 (link)), with primers grouped as previously described (35 (link)). PCR products were analyzed on 1% 96-well ethidium bromide–stained precast gels (Embi Tec). PCR-amplified V_H and V_L genes were purified and sequenced. Sequences were analyzed, and V(D)J arrangements were inferred using IMGT/V-Quest (www.imgt.org).