G50 microspin column

0.5 μg of bacteriophage Lambda DNA (New England Biolabs) was labeled with the Mirus Label-IT rhodamine TM reagent (Mirus Bio) according to the manufacturer’s recommendations with the exception that four-fold less labeling reagent was used. The DNA was then processed through a G50 MicroSpin column (GE Healthcare) to remove excess label.

ADCP assays were conducted using virus labeled with a pH-sensitive dye called pHrodo (as has been used to study viral entry in previous studies, ref. 51 (link)). Purified CHIKV virus was incubated for 45 minutes at room temperature with 50 μM pHrodo Red, succinimidyl ester (Thermo Fisher Scientific, P36600) in 0.1 M bicarbonate buffer. Excess dye was removed by passing labeled virus through a G-50 micro-spin column (GE Healthcare). Labeled CHIK virus was incubated with serum samples (diluted at 1:100 in cell culture media) for 1 hour at 37°C under 5% CO₂. Virus-antibody complexes were then added to a human monocytic cell line, THP-1 (ATCC TIB-202), and incubated for 5 hours at 37°C under 5% CO₂. Following incubation, cells were washed with 1X PBS, fixed with 4% PFA, and resuspended in FACS buffer (2% FBS, 0.4% 0.5 M EDTA in 1X PBS). The percentage and MFI of virus⁺ cells were assessed using flow cytometry (BD LSR Fortessa). Representative FACS plots for ADCP with no immune sera and with MV-CHIK–vaccinated immune sera are presented in Supplemental Figure 6A. Phagocytic scores were calculated by multiplying the percentage of cells positive for viral entry with the MFI of those positive cells. Normalized phagocytic scores were calculated by dividing ADCP phagocytic scores by log₁₀-transformed CHIKV-specific IgG endpoint titers.