Anti guinea pig complement c3 fitc

ADCD was adapted from methods described previously (76 (link)). Briefly, SARS-CoV-2 S-expressing expi293F cells were incubated with 10-fold diluted, heat-inactivated (56 °C for 30 min) plasma samples for 30 min at 37 °C. Cells were washed twice and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS (R10 media). During this time, lyophilized guinea pig complement (CL4051, Cedarlane) was reconstituted in 1 mL of cold water and centrifuged for 5 min at 4 °C to remove aggregates. Cells were washed with PBS and resuspended in 200 μL of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca²⁺ and Mg²⁺ (IBB-300X, Boston BioProducts). After incubation at 37 °C for 20 min, cells were washed in PBS 15 mM (ethylenedinitrilo)tetraacetic acid (ThermoFisher Scientific) and stained with an anti-Guinea Pig Complement C3 FITC (polyclonal, ThermoFisher Scientific). Cells were then fixed with 4% formaldehyde solution, and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience).

SARS-CoV-2 spike-expressing expi293F cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 Spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank, ACC# MN988713) or B.1.1.529. Stable transfectants were single-cell–sorted and selected to obtain a high-level spike surface expressing clone. An ADCD assay was adapted from (38 (link)). Briefly, 293F-Spike–expressing cells were incubated with 10-fold diluted heat-inactivated (56°C for 30 min) plasma samples for 30 min at 37°C. Cells were washed twice and resuspended in RPMI with 10% FBS. Lyophilized guinea pig complement (CL4051, Cedarlane, Burlington, Canada) was reconstituted per the manufacturer’s instructions in 1 ml of cold water and centrifuged at 13,000 rpm for 5 min at 4°C. Cells were washed with PBS and resuspended in 200 μl of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca²⁺ and Mg²⁺ (IBB-300x, Boston BioProducts, Ashland, MA). After incubation at 37°C for 20 min, cells were washed in PBS with 15 mM EDTA (Thermo Fisher Scientific) and stained with an anti–guinea pig complement C3 FITC (polyclonal, Thermo Fisher Scientific). Cells were fixed with 4% formaldehyde solution, and fluorescence was evaluated on an LSR II (BD Biosciences).