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.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.