1300exi cytometer

The ADCD assay was adapted from Fischinger et al.^{84 (link)}. Antigen was coupled to non-fluorescent Neutravidin 1 μm beads (Invitrogen, #F8777) as described for ADCP. Immune complexes were formed by incubating 10 μL of coupled beads with 10 μL of diluted sample for 2 h at 37 ˚C. Plates were spun down, and immune complexes were washed with PBS. Lyophilized guinea pig complement (Cedarlane, #CL4051) was resuspended in cold water, diluted in Gelatin Veronal Buffer, Boston BioProducts, IBB-290X) and added to the immune complexes. The plates were incubated for 50 min at 37 ˚C and the reaction was stopped by washing the plates twice with 15 mM EDTA in PBS. To detect complement deposition, plates were incubated with Fluorescein-conjugated goat anti-guinea pig complement C3 (1:50 diluion) (MP Biomedicals, #0855385) for 15 min in the dark. Fluorescence was acquired with a Stratedigm 1300EXi cytometer.

The ADCP assay was adapted from (39 (link)). Briefly, gp140 SHIV_SF162p3 and the recombinant Mymetics gp41 antigens were biotinylated using sulfo-NHS LC-LC biotin, coupled to yellow-green, fluorescent Neutravidin 1 μm beads (Invitrogen, F8776) for 2 h at 37°C and washed two times in 0.1% bovine serum albumin (BSA) in PBS. Ten μl/well of coupled beads were added to 96-well plates with 10 μl/well of diluted sample for 2 h at 37°C to form immune complexes. After incubation, the immune complexes were spun down, and supernatants were removed. THP-1 cells were added at a concentration of 2.5 x 10⁴ cells/well and incubated for 18 h at 37°C. After incubation, the plates were spun down, the supernatant was removed, and cells were fixed with 4% paraformaldehyde (PFA) for 10 min. Fluorescence was acquired with a Stratedigm 1300EXi cytometer. Phagocytic score was calculated using the following formula: (percentage of FITC+ cells) * (the geometric mean fluorescent intensity (gMFI) of the FITC+ cells)/10,000. A polyclonal HIVIg pool available from the NIH AIDS Reagent Program was used as a positive control. Serum from a human HIV-seronegative donor was used as negative control.

The ADNP assay was adapted from Karsten et al. (40 (link)); gp140 SHIV_SF162p3 and the recombinant Mymetics rgp41 antigens were coupled to beads and immune complexes were formed as described for ADCP. Neutrophils were isolated from fresh whole ACD-anticoagulated blood using EasySep Direct Human Neutrophil Isolation kit (Stem Cell, 19666), resuspended in R10 medium, and added to plates at a concentration of 5x10⁴ cells/well. The plates were incubated for 30 min at 37°C. The neutrophil marker CD66b (Pacific Blue conjugated anti-CD66b; BioLegend, 305112) was used to stain cells. Cells were fixed for 10 min in 4% PFA. Fluorescence was acquired with a Stratedigm 1300EXi cytometer and phagocytic score was calculated as described for ADCP.