Generation of K46 B cell lines expressing PGT121, PGT145, or VRCO1 has been previously described (92). Briefly, K46 cells expressing a doxycycline-inducible form of bNAb BCRs were maintained in advanced Dulbecco’s modified Eagle’s medium (DMEM) (Gibco), supplemented with 10% fetal calf serum, penicillin/streptomycin antibiotics, and puromycin (2 μg/ml; Gibco). Cells were treated overnight in doxycycline (1 μg/ml; Clontech) to induce human BCR expression. After loading with Indo-1 (Molecular Probes) at 1 μM for 1 hour at 37°C, washed cells were stimulated with the indicated agents at a concentration of 10 μg ml−1: anti-mouse IgM (Jackson ImmunoResearch), UFO-BG or an HR1-redesigned gp140 trimer (36) with a T helper epitope (PADRE) fused to the C terminus, and UFO-BG-FR or I3-01 nanoparticle presenting an HR1-redesigned gp140 trimer. Calcium mobilization was assessed on an LSR II flow cytometer (BD Biosciences). In each run, the unstimulated B cells were first recorded for 60 s; immunogen was added, mixed thoroughly, and recorded for 180 s; and then 1 μl of ionomycin (1 μg ml−1; Sigma) was added and recorded for another 60 s to verify Indo loading.

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