Neutravidin fluospheres beads

To examine ADP mediated by COVID-19 convalescent plasma, a previously published bead-based ADP assay was adapted for use in the context of SARS-CoV-2²⁴. SARS-CoV-2 S trimer was biotinylated using EZ-Link Sulfo-NHS-LC biotinylation kit (Thermo Scientific) with 20mmol excess according to manufacturer’s instructions and buffer exchanged using 30kDa Amicon centrifugal filters (EMD millipore) to remove free biotin. The binding sites of 1 μm fluorescent NeutrAvidin Fluospheres beads (Invitrogen) were coated with biotinylated S at a 1:3 ratio overnight at 4°C. S-conjugated beads were washed four times with 2% BSA/PBS to remove excess antigen and incubated with plasma (1:100 dilution) for 2 hours at 37°C in a 96-well U-bottom plate (see Figure S5 for optimization). THP-1 monocytes (10,000/well) were then added to opsonized beads and incubated for 16 hours under cell culture conditions. Cells were fixed with 2% formaldehyde and acquired on a BD LSR Fortessa with a HTS. The data was analyzed using FlowJo 10.7.1 (see Figure S4 for gating strategy) and a phagocytosis score was calculated as previously described⁵³ (link) using the formula: (%bead-positive cells × mean fluorescent intensity)/10³. To account for non-specific uptake of S-conjugated beads, the phagocytosis scores for each plasma sample were subtracted with that of the “no plasma” control.