*2.8. Flow Cytometry Analysis*

The expression of NS1 protein in infected HMC-1 cells was analyzed by flow cytometry. Cells were collected by centrifugation, and suspended in PBS for 30 min, 6 h or 24 h after infection with di fferent MOIs. Approximately 10<sup>6</sup> cells/well were fixed in 4% formaldehyde for 25 min and permeabilized with 0.05% saponin for 30 min. Next, cells were incubated with a 1:1000 dilution of the mouse monoclonal IgG antibody against ZIKV non-structural protein NS1 (Arigo Biolaboratories, Taiwan, Republic of China) for 1 h at 37 ◦C before being washed with PBS. This was followed by an incubation with a 1:200 dilution of an Alexa 488-conjugated anti-mouse (Thermo Fisher, Waltham, MA, USA) for 30 min. After washing with PBS, cells were suspended in PBS and applied to a flow cytometer (Facs Calibur; BD Biosciences, San Jose, CA, USA) to measure fluorescence, which was analyzed o ffline with Summit 6.1 software.

## *2.9. Measurement of Mast Cell Degranulation*

Mast cell degranulation was evaluated by measuring the activity of the granule-stored enzymeβ-hexosaminidase that was secreted into the extracellular medium. Cells were infected with MOI 0.1, 0.2 or 1 in 6-well plates (1 × 10<sup>6</sup>/well) for 30 min. Aliquots of the supernatant (15 μl) were transferred to 96-well plates and incubated with 60 μL of substrate (1 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide) in 0.05 M sodium citrate (pH 4.5) for 60 min at 37 ◦C. In addition, we used 60 μL of substrate solution (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma, St. Louis, MS, USA) in 100 mM sodium citrate, pH 4.5) and incubated for 60 min at 37 ◦C. Reactions were stopped by adding 150 μL of 0.1 M Na2CO3-NaHCO3 bu ffer (pH 10). Enzyme activity was measured as the absorbance at 405 nm. Total β-hexosaminidase activity was determined by releasing all enzyme through lysis with 0.1% Triton X-100 and measuring activity from a 15 μl aliquot. As a positive control for degranulation, we used 20 μg/mL of 48/80 compound (Sigma, St. Louis, MS, USA). The results are presented as the percentage of total β-hexosaminidase content of the cells.
