Zombie violet fixable viability stain

Following viral infection, moDCs were collected, stained with Zombie Violet™ Fixable Viability stain (BioLegend), washed, fixed, and permeabilized using BD Cytofix/Cytoperm reagents, and then intracellularly stained with FITC- or AF647-conjugated 4G2 mAb. Cells were washed twice with BD Perm/Wash Buffer and resuspended in FACS buffer. To quantify neutral lipid content, moDCs were collected, stained with BODIPY 493/503, and then treated as described above. The percentage uninfected and infected cells and the neutral lipid content in uninfected and infected cells were quantified by flow cytometry using a LSRII flow cytometer (BD Biosciences) or MA900 cell sorter (Sony) and Flowjo 10.8 software.

moDCs were infected with ZIKV or DENV2 for 24 h at MOIs of 0.5 and 1, respectively. Huh7.5 cells and NPCs were infected with ZIKV for 24 h at an MOI of 1. The cells were then collected, incubated with Zombie Violet™ Fixable Viability stain (BioLegend) for 20 min at 4 °C, washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin in the presence of RNasin ribonuclease inhibitor (400 U/ml). The cells were centrifuged, resuspended in wash buffer (PBS containing 0.2% bovine serum albumin [BSA], 0.1% saponin, and 400 U/ml RNasin), and incubated for 5 min with human Fc Blocker in staining buffer (1% BSA, 0.1% saponin, 1600 U/ml RNasin) at 4 °C. The cells were then incubated with Alexa Fluor 647-conjugated 4G2 (anti-flavivirus group antigen), incubated for 30 min at 4 °C, and centrifuged at 1000×g for 3 min at 4 °C. The cells were washed in wash buffer, re-centrifuged, and finally resuspended in sort buffer (PBS containing 0.5% BSA and 1600 U/ml of RNasin). ZIKV+, ZIKV−, DENV+, and DENV− cells were sorted using a FACSAria (BD Biosciences) or MA900 (Sony) sorter.

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.