Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

In vitro proliferation assays Naive mature B cells were purified and labeled with CTV as described for adoptive transfer experiments. Cells were adjusted to a concentration of 2 × 10 6 cells/ml in RPMI (Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific), 0.1 mg/ml streptomycin, 2 mM L-glutamine (Thermo Fisher Scientific), 0.02 mg/ml gentamicin (Thermo Fisher Scientific), and 0.027.5 mM 2-mercaptoethanol (Sigma-Aldrich). 2 × 10 5 cells from each sample were added per well of a 96-well flat bottomed plate, and cells were cultured in the presence or absence of 2, 5, 10, or 25 µg/ml F(ab9) 2 goat anti-mouse Ig (Jackson ImmunoResearch) in a final volume of 0.2 ml and incubated for 72 h at 37°C before analysis by flow cytometry.