Harmony high content imaging and analysis software version 4

We kept fixed and immunostained C3H mouse feeder cells in 200 µl PBS (Sigma Aldrich) or 200 µl benzyl-alcohol/benzyl benzoate (BABB) (Sigma-Aldrich) per well of an organic solvent-resistant 96-well flat-bottom COC plate and detected the fluorescence emission within set emission bands with the high content microscopy system Operetta (PerkinElmer). Further analysis was performed via the Harmony High Content Imaging and Analysis Software Version 4.1 (PerkinElmer)⁵³ .

At varying time points post-infection, the mock and DENV-infected Vero cells in black 96-well plates were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, and permeabilized with 0.2% Triton-X-100 in PBS for 10 min at RT. Thereafter, the cells were successively incubated at RT with a mouse anti-NS1 monoclonal antibody (clone 1A4) for 1 h and with a mixture of Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:1000 dilution), Hoechst 33342 (1:1000 dilution; Molecular Probes, Eugene, OR, USA), and HCS CellMask Deep Red Stain (1:50,000 dilution; Invitrogen) for 30 min in the dark. Three washes with PBS were performed after the primary and secondary staining steps. Images of the stained cells were captured with a 20× objective lens for 25 fields in each sample and analyzed by an Operetta High Content Imaging System using Harmony high-content imaging and analysis software version 4.1 (PerkinElmer, Inc., Waltham, MA, USA) to assess the proportion of DENV-infected cells and the relative levels of DENV NS1 expression.