Alexafluor 647 conjugated goat anti human igg h l ab

Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) were transfected into 2 × 10⁶ 293T cells. To determine the Hill coefficients, cells were preincubated with increasing concentrations of soluble ACE2 (0 to 11,500 nM), ACE2-Fc (0 to 500 nM), or the monoclonal antibody CR3022 (0 to 270 nM) 48 h post-transfection. sACE2 binding was detected using a polyclonal goat anti-ACE2 (RND systems). AlexaFluor-647-conjugated goat anti-human IgG (H+L) Ab (Invitrogen) and AlexaFluor-647-conjugated donkey anti-goat IgG (H+L) Ab (Invitrogen) were used as secondary antibodies. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on an LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA). Hill coefficient analyses were done using GraphPad Prism version 8.0.1 (GraphPad, San Diego, CA, USA).

Using the standard calcium phosphate method, 10 µg of spike expressor and 2 µg of a green fluorescent protein (GFP) expressor (pIRES2-GFP, Clontech) were transfected into 2 × 10⁶ 293T cells. At 48 h post transfection, 293T cells were stained with anti-Spike monoclonal antibodies CV3-25 (5 µg/mL) or using the ACE2-Fc chimeric protein (20 µg/mL) for 45 min at 37 °C, 22 °C, or 4 °C. Alternatively, to determine the Hill coefficients, cells were preincubated with increasing concentrations of sACE2 (0 to 665 nM) at 37 °C or 4 °C. sACE2 binding was detected using a polyclonal goat anti-ACE2 (RND systems, Minneapolis, MN, USA). AlexaFluor-647-conjugated goat anti-human IgG (H + L) Ab (Invitrogen) and AlexaFluor-647-conjugated donkey anti-goat IgG (H + L) Ab (Invitrogen) were used as secondary antibodies to stain cells for 30 min at room temperature. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on an LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo v10.3 (Tree Star, Ashland, OR, USA). Hill coefficient analyses were done using GraphPad Prism version 8.0.1 (GraphPad, San Diego, CA, USA).

SARS-CoV-2 authentic viruses (D614G or B.1.1.7 (α) variant) were used to infect the pAECs at a multiplicity of infection (MOI) of 0.1. Forty-eight hours after infection, cells were detached by PBS-EDTA (10 mM) and 0.2 × 10⁶ cells per sample were stained with CV3-25 (5 µg/mL) or plasma (1/1000) for 30 min at 37 °C. Alexa Fluor-647-conjugated goat anti-human IgG (H + L) Ab (1/1000, Invitrogen, Waltham, MA, USA.) was used as a secondary antibody to stain the cells for 30 min at room temperature. The cells were then fixed with PBS containing 4% paraformaldehyde for 48 h at 4 °C. Then, the cells were stained intracellularly for SARS-CoV-2 nucleocapsid (N) antigen, using the Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences) and 1 µg/mL anti-N mAb (clone mBG17; Kerafast, Boston, MA, USA) conjugated with the Alexa Fluor 488 dye according to the manufacturer’s instructions (Invitrogen). The percentage of infected cells (N+ cells) was determined by gating the living cell population based on the viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on a LSR II cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data analysis was performed using FlowJo v10.5.3 (BD Biosciences).