Igg pe cy7

PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.

In order to investigate targeting ability of the antibody in tissue condition, we first performed near-infrared fluorescent imaging on excised aortas. Briefly, anti-CD11b-PE-Cy7 (BD Bioscience) was injected to 5 ApoE^−/− and 5 C57 mice via tail vein (1 mg/kg), while IgG-PE-Cy7 (BD Bioscience) with same dose was injected to another 5 ApoE^−/− mice. Twenty four hours later, mice were sacrificed. NaCl (0.9%, 5 mL) and paraformaldehyde (4%, 5 mL) were sequentially injected into vascular system to eject blood and fix vessels. Then, aorta was carefully dissected, placed on a microscope slide, covered with gauze (rinsed with 0.01 M PBS), and imaged within 1 h in the Carestream FX PRO (Kodak, USA). The Cy7 label was excited using a wavelength filter of 615–665 nm, while the emission was recorded using a 695–770 nm filter. Fluorescent images were acquired with identical exposure time for all aortas. Area percentage of plaque was measured and total photon count was recorded around plaque areas. Mean signal intensity was calculated by dividing the total photon count to the total area of plaques (SI-mean). Likewise, noise intensity (NI-mean) was measured by drawing a ROI in normal area of the aorta. Finally, a signal-to-noise ratio (SNR) was calculated as: SI-mean/NI-mean.

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).