Anti cd19 buv805

Cryopreserved PBMC from VRC 018 clinical trial (ClinicalTrials.govNCT03783130)^{22 (link)} at 2 weeks after third boost time point were thawed, treated with Benzonase nuclease (Millipore Corp.), washed with PBS and stained with viability dye Aqua Fluorescent Reactive (Invitrogen) for 2 min followed by staining with the following human antibodies: anti-IgM-BB700 (BD, customized), anti-CD21-PE594 (BD Biosciences, 563474), anti-CD20-APCH7 (BD Biosciences, 560734), anti-IgG-BUV3950 (BD Biosciences, 564229), anti-CD3-BV510 (BD Biosciences, 740187), anti-CD14-BV510 (Biolegend, 301842), anti-CD56-BV510 (BD Biosciences, 740171), anti-CD38-BUV661 (BD Biosciences, 612969), anti-CD19-BUV805(BD Biosciences, 749173), anti-IgD-BV570 (BD Biosciences, 624298), anti-CD27-BV605 (Biolegend, 302830) along with BG505-AF488, glycan-base BG505-PE and glycan-base BG505-APC probes diluted in Brilliant Stain Buffer (BD Biosciences, 563794) for 30 min at 4°C protected from light. Cells were washed with PBS containing 0.1% BSA twice and analyzed on FACS Symphony A5 (BD Biosciences) using Diva software. Cells were gated on live singlets CD3⁻ CD4⁻ CD14⁻ CD56⁻ IgD⁺ IgM⁺ CD19⁺ CD20⁺ IgG⁺ memory B cells and all glycan-base BG505 positive cells were single-cell sorted in 96-well plates coated with 5 μL of TCL buffer (Qiagen) containing 1% of 2-Mercaptoethanol (Sigma-Aldrich).