Goat anti guinea pig c3 fitc

4 × 10⁶ (ACE2)-CHO cells were pulsed with 20 μg biotinylated SARS-CoV-2 (COVID-19) S1 and RBD proteins (Acro Biosystems) for 30 min at 37°C. Excess, unbound antigens were removed by washing cells once with complete medium. Heat-inactivated monkey plasma (10 μl) were added to the antigen-pulsed cells and incubated for another 30 min at 37°C. Freshly resuspended lyophilized guinea pig complement (Cedarlane) diluted 1:20 with veronal buffer 0.1% gelatin with calcium and magnesium (Boston BioProducts) were added to the cells for 2 h at 37°C. Following a wash with 1X PBS, cells were assessed for complement deposition by staining with goat anti-guinea pig C3-FITC (MP biomedicals). After fixing, cells were analyzed by flow cytometry and ADCD are reported as MFI of FITC+ cells.

ADCD was assessed using a flow cytometry-based complement-fixing assay^{109 (link)}. Briefly, 10 × 10⁶ HUT78cells (obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: HUT 78 Cells, ARP-89, contributed by Dr. Adi Gazdar and Dr. Robert C. Gallo) were pulsed with 2 µg of recombinant HIV-1 gp120 from strain CN54 (Acro Biosystems; catalog# GP0-V182E6) for 20 min at 37 °C. Unbound gp120 was removed by washing the cells twice with 1% PBS-BSA buffer. Bulk IgG samples from study participants were added to the antigen-pulsed cells and incubated for another 30 min at 37 °C. Freshly resuspended lyophilized guinea pig complement (Cedarlane Labs; catalog# CL4051), diluted 1:20 with veronal buffer 0.1% gelatin with calcium and magnesium (Boston BioProducts; catalog# IBB-300), was added to the cells for 2 h at 37 °C. After washing with 1X PBS, the cells were assessed for complement deposition by staining with goat anti-guinea pig C3-FITC (MP Biomedicals; catalog# 0855385). After fixing, the cells were analyzed by flow cytometry, and ADCD was reported as the mean fluorescence intensity (MFI) of FITC-positive cells. A gating example is shown in Supplementary Fig. 9c.