Formaldehyde solution

SARS-CoV-2 spike-expressing FreeStyle™ 293-F (RRID:CVCL_D603; Invitrogen cat. no. R79007) cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein (GenScript) matching the amino acid sequence having mutations E156G, D614G, P681R, T19R, T478K, L452R, D950N, and 157–158 del for Delta. Stable transfectants were single-cell sorted and selected to obtain a high-level Spike surface expressing clone (293F-Spike-Delta). 293F-Spike-Delta cells were incubated with 100 μl of 4-fold serial dilutions of plasma starting at 100-fold for 30 min at 4 °C. Cells were washed twice and stained with anti-human IgG PE, anti-human IgM Alexa Fluor 647, and anti-human IgA FITC (Southern Biotech, Birmingham, AL, USA, cat. nos. 2040-09, 2020-31, and 2050-02). Cells were then fixed with 4% formaldehyde solution (Tousimis, Rockville, MD, USA, cat. no. 1008B) and fluorescence was evaluated on a LSRII (BD Bioscience).

This assay was described previously.²⁷ Briefly, 293F-Spike-Delta cells, described above, were incubated with 100 μl of diluted plasma (10-fold) for 30 min at 4 °C. Cells were washed twice and resuspended in R10 media. Lyophilized guinea pig complement (CL4051, Cedarlane, Burlington, Canada, cat. no. CL4051) was reconstituted per the manufacturer’s instructions in 1 mL cold water and centrifuged to remove aggregates 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⁺ (Sigma-Aldrich, St. Louis, MO, USA, cat. no. G6514). After incubation at 37 °C for 20 min, cells were washed in PBS 15 mM EDTA (ThermoFisher Scientific Baltics UAB, Vilnius, Lithuania, cat. no. AM9260G) and stained with an anti-guinea pig complement C3 FITC (polyclonal, MPBiomedicals, Solon, OH, USA, cat. no. 0855385). Cells were fixed with 4% formaldehyde solution (Tousimis, Rockville, MD, USA, cat. no. 1008B) and fluorescence was evaluated on a LSRII (BD Bioscience).

This assay was described previously.²⁶ (link) Briefly, biotinylated SARS-CoV-2 Delta spike trimer (ECD; SinoBiological, Beijing, China, cat. no. 40589-V08H10) was incubated with yellow-green neutravidin-fluorescent beads (Molecular Probes, Eugene, OR, USA, cat. no. F8776) for 2 h at 37 °C. 10 μl of a 100-fold dilution of beads–protein was incubated 2 h at 37 °C with 100 μl of diluted plasma (900-fold) before addition of 25,000 cells per well THP-1 cells (RRID:CVCL_0006; Millipore Sigma, Burlington, MA, USA, cat. no. 88081201). After 19 h incubation at 37 °C, the cells were fixed with 2% formaldehyde solution (Tousimis, Rockville MD USA, cat. no. 1008B) and fluorescence was evaluated on a LSRII (BD Bioscience). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean fluorescence intensity (MFI) of the bead-positive cells and dividing by 10⁴.