Igg pe cy7 g18 145

Antigen specificity of (gl-)AR3C and (gl-)HEPC74 Ramos B cells to H77 E2E1 and UKNP4.1.1 E2E1 was detected using labeled probes and flow cytometry as previously^{42 (link),52 (link)}. Briefly, biotinylated H77 and UKNP4.1.1 E2E1-foldon-Avi-His were individually multimerized with fluorescently labeled AF647 (Biolegend) and BV421 (Biolegend) streptavidin at a 2:1 protein to fluorochrome molar ratio and incubated for 1 h at 4 °C. Unbound streptavidin conjugates were quenched with 10 µM biotin (Genecopoiea) for 15 min. The antigen-probe cocktails were then used to stain 5 × 10⁵ cells together with live/DEAD dye (eBioscience™ Fixable Viability Dye eFluor™ 780, Thermo Fisher), IgG PE-Cy7 (G18-145, BD Biosciences) in FACS buffer (PBS supplemented with 1 mM EDTA and 2% fetal calf serum). The live/DEAD marker was titrated for signal-to-noise ratio and IgG PE-Cy7 was used in a dilution of 2.5 μL in 50 μL. Stained samples were subsequently washed twice with FACS buffer and acquired on the BD LSRFortessa^TM for cell analysis. Analysis was performed using FlowJo v10.8.1. Ramos cells were first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Live cells were selected and subsequently gated on GFP+ and IgG+. Antigen-specific Ramos B cells were double positive for the HCV H77 and 4.1.1 E2E1- foldon-Avi-His probes (AF647 and BV421).

HA-trimer specific B cell response from six healthy human donors was analyzed by flow cytometry. Biotinylated recombinant proteins were conjugated with streptavidin fluorophores resulting in fluorescent labelled-probes. HA-trimers, both WT and RBS-mut, trimeric-stem and monomeric-head were conjugated in an 8:1 ratio (w/w) to streptavidin-conjugates BV421 (0.1 mg/mL, BioLegend, Amsterdam, The Netherlands), BB515 (0.1 mg/mL, BD, Vianen, The Netherlands), or AF647 (0.5 mg/mL, BioLegend) at 4 °C, for 1–2 h.
PBMCs were thawed and stained with the following surface markers: CD3-v500 (UCHT1, BD), CD20-PE-CF594 (2H7, BD), IgM-BV605 (MHM-88, BioLegend), IgG-PE-Cy7 (G18-145, BD), IgA-PE (polyclonal, Southern Biotech), the live/dead marker (viability-eF780, Invitrogen) and streptavidin-conjugated probes at 4 °C, for 30–60 min. Flow cytometry was performed on an ARIA 4 lasers (BD) flow cytometer and the data analysis was performed with FlowJo v10.6.
Live, CD3-, CD20+ and HA-trimer+ cells were single cell sorted into a 96-well plate that contained 20 µL lysis buffer consisting of 20 U RNAse inhibitor (Invitrogen), first strand superscript III buffer (Invitrogen) and 1.25 µL of 0.1 M DTT (Invitrogen). The plates with the sorted cells were stored at −80 °C for at least 24 h before performing the RT-PCR to obtain cDNA.