Human trustain fcx blocker

Raji‐Env and Raji‐Ctr cells (1 × 10⁵) were washed in PBS containing 2% HI‐FBS, and pre‐incubated with 5 μL of Human TruStain FcX blocker (BioLegend) in 100 μl staining volume for 5 min at RT. Subsequently, the cells were incubated with or without 30 μg/ml of anti‐HIV antibodies for 45 min at 4°C. Cells were prepared for flow cytometry by washing once with PBS containing 2% HI‐FBS followed by incubation with 5 μl of Brilliant Violet 421 anti‐human IgG Fc Antibody (clone M1310G05) (Biolegend) or 0.015 μg of Brilliant Violet 421 Streptavidin (BioLegend) in 100 μl staining volume for 20 min at 4°C. The gating strategy for flow cytometry analysis is shown in Fig EV5.

Raji‐Env and Raji‐Ctr cells (5 × 10⁴) were resuspended in 100 μl RPMI containing 25% NHS from healthy donors and 100 U/ml P/S. Cells were seeded in a 96‐well culture plate and incubated for 1 and 24 h at 37°C with or without 10 μg/ml antibody or BiCE (unless otherwise stated), and with or without a C3 nanobody inhibitor (hC3Nb2) using C3:hC3Nb2 molar ratio of 1:4. HC3Nb2 has previously been described by H. Pedersen and GR Andersen (Pedersen et al, 2020 (link)). After 1 and 24 h, the cells were prepared for flow cytometry. To measure CDC, cells were stained with 1 μl of the live/dead marker Zombie Violet Fixable Viability Stain (Biolegend) in 100 μl staining volume for 30 min at 4°C. Complement‐dependent cytotoxicity was determined using the following formula: 100 × (% of dead cells with antibody − % of dead cells without antibody) / (100 − % of dead cells without antibody). Negative values were set to zero. Second, to measure complement deposition, cells were pre‐incubated with 5 μl of Human TruStain FcX blocker (BioLegend) in 100 μl staining volume for 5 min at RT, followed by incubation with 5 μl of APC anti‐complement C3b/iC3b Antibody (clone 3E7/C3b) (BioLegend) or 5 μl of APC Mouse IgG1, κ Isotype Ctrl Antibody (clone MOPC‐21) (BioLegend) in 100 μl staining volume for 20 min at 4°C. The gating strategy for flow cytometry analysis is shown in Fig EV5.