Dylight antibody labeling kit

The primary and secondary antibodies used in this study are described in Table S1. IL-1β (PeproTech, Rocky Hill, NJ, USA) stimulation of pMBMEC monolayers was done for 16–20 h at a concentration of 0.05 ng/ml (IL-1 β^lo) or 20 ng/ml (IL-1 β^hi). Angubindin-1, used to detect LSR/angulin-1, was coupled with DyLight 550 NHS ester with the DyLight Antibody labeling kit (Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer's instructions.

Cells were harvested and treated with 1 μg/ml of anti-podoplanin antibodies or control mouse IgG (Sigma-Aldrich, St Louis, MO, USA), following incubation with 4 μg/mL of Alexa Flour 488-conjugated anti-mouse IgG (H + L) (Thermo Fisher Scientific, Waltham, MA, USA). For CLEC-2-binding analysis, cells were incubated with 0.4 μg/mL of (His)₁₀-tagged human CLEC-2 protein (R & D Systems, Minneapolis, MN, USA), following incubation with 1/500 dilutions of Alexa Flour 488-conjugated anti-penta-His antibody (QIAGEN, Venlo, Netherlands). Flow cytometric analyses were performed using Cytomics FC500 flow cytometry system (Beckman Coulter, CA, USA). In some experiments, MS-1 and PG4D2 mAbs were used as primary antibodies, following the conjugation of DyLight594 using the DyLight antibody labeling kit (Thermo Fisher Scientific, Waltham, MA, USA).

The binding kinetics against CLDN3 on the cell surface were measured using LigandTracer Green (Ridgeview Instruments AB, Vänge, Sweden). The hCLDN3/HEK293, hCLDN3/TOV-112D, and mCLDN3/HEK293 cells, as positive cells, and HEK293 and TOV-112D cells, as negative cells, were seeded on a limited area of 100 mm culture dish at a density of 3 × 10⁵ cells/mL in 500 μL culture medium, and after 6 h, 10 mL growth medium was added to culture dish. Cells were incubated overnight, and 3 mL of the medium was changed before the experiment. The h4G3 was labeled with DyLight dye 488 using DyLight Antibody Labeling Kits (Thermo Fisher Scientific) following the manufacturer’s instructions. The cell culture dish was clamped onto the device and the fluorescence baseline was recorded. Each time the respective fluorescence reached equilibrium, Dylight dye 488-labeled h4G3 was added stepwise to a final concentration of 3 nM and 9 nM for hCLDN3 cell lines, and 30 nM and 90 nM for mCLDN3 cell lines. In the dissociation phase, the remaining medium was removed, and 3 mL fresh medium was added to the culture dish. All measurements were performed using a 15 s detection time and 4 s detection delay. Recorded data were analyzed by TraceDrawer (Ridgeview Instruments AB).

Quadrivalent FluMos-v1, monovalent, bivalent, and trivalent nanoparticles^{39 (link)}, HA-trimer, Pentamer, and their formulation buffers were produced/prepared at the VPP. Monoclonal antibodies m-H1, m-H3, m-HBv, and m-HBy (1B01) were obtained from the VRC, and another m-Hby was purchased from Sino Biological (Catalog number 11053-MM09). Dylight fluorescent labeling kits were purchased from Thermo Scientific (DyLight Antibody Labeling Kits, Thermo PI53024). CF405-S Fluorescent labeling kit was purchased from Biotium (Catalog number 92211). FabALACTICA Fab kits were purchased from Genovis (AK-AFK-025). Amicon Ultra centrifugal filters were purchased from Millipore (Ultracel 100k, UFC510096). Sodium phosphate monobasic (monohydrate) and sodium phosphate dibasic (heptahydrate) was purchased from JT Baker (Catalog number 3802-01, and 3803-01, respectively).

Monoclonal antibody CR3022 was produced by transient transfection of 293 F cells with cDNA expression plasmids obtained from BEI resources. The cells were transfected at a density of approximately 2 × 10⁶ cells/ml with equal amounts of each plasmid using Expifectamine reagent (Thermo Fisher) according to the instructions provided by the manufacturer. The supernatant was harvested after 4–5 days and antibody purified on Protein G Sepharose (Abcam, GE Healthcare) following standard protocols. Monoclonal antibodies I1 and I14 against the VSV glycoprotein and monoclonal antibody 23H12 against the VSV matrix protein were purified from hybridoma supernatant by affinity chromatography on Protein G sepharose. The antibodies were labeled with fluorophores using Dylight antibody labeling kits from Thermo Fisher. The following SARS-CoV-2-specific human monoclonal antibodies were kindly provided by Distributed Bio: DB_A03-09, 12; DB_B01-04, B07-10, 12; DB_C01-05, 07, 09, 10; DB_D01, 02; DB_E01-04, 06, 07; DB_F02-03.