Facslyric machine

At specified time points, cells were harvested and were washed in 2% fetal calf serum (FCS) in PBS. Immunostaining was performed using a 1:100 dilution of an in-house-generated mouse anti-HMPV F hybridoma antibody followed by a 1:100 dilution of a secondary polyclonal rabbit anti-mouse immunoglobulin G (IgG)-fluorescein isothiocyanate (FITC) antibody (Dako). Cells were fixed with 2% paraformaldehyde (PFA) in PBS, and the percentage of cells infected with HMPV was analyzed using a FACSLyric machine (BD Biosciences). IFN levels were quantified by a firefly luciferase reporter assay as described previously (44 (link)).

In some ADE experiments, flow-cytometry was used as a read-out to determine the percentage of infected cells. For this, cells were collected on day two post infection and stained with live/dead stain. Cells were blocked with 10% normal goat serum (NGS, Dako, Glostrup, Denmark) and total human Fc block (BD Biosciences). After fixation and permeabilization using BD Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ, USA), intracellular staining for ZIKV E-protein was performed with a mouse 4G2 antibody (MAB10216, clone D1-4G2-4-15; Millipore, Darmstadt, Germany) followed by an APC/Cy-7 conjugated goat anti-mouse IgG2a secondary antibody (Abcam, Cambridge, UK). Flow cytometry was performed with the FACS Lyric machine (BD Biosciences, USA). Data were analyzed using FlowJo 10.6.1 software (Ashland, OR, USA). All experiments were performed three times (biological replicates), and each experiment included duplicate (technical replicates) measurements from which the average was calculated and used for further analysis. To demonstrate that intracellular ZIKV-E staining represents productive infection and not just phagocytosis, some ZIKV-infected cells were also stained with an anti-DENV NS3 antibody (E1D8, My Biosource, San Diego, CA, USA), which has been shown to cross-react with ZIKV NS3 [29 (link)].