Analyzing DENV Infection and Immunomodulation

Flow cytometry was used to assess the frequency of DENV-infected cells, expression levels of VDR and, changes of TLR3/4/9 expression, as previously described [17 (link),18 (link)]. Flow cytometry was also used for assessing the production of ROS production by the detection of the dihydrorhodamine 123 (DHR 123; Sigma Aldrich, USA). DENV infection was evaluated through the intracellular detection of DENV E antigen at the indicated time points and the cells were fixed using a fixation/permeabilization buffer (eBioscience, USA). Following washing steps with PBS, cells were stained with the monoclonal antibody, 4G2 (Millipore, Germany) for 40 min, followed by 40 min staining with goat anti-mouse IgG-FITC (Thermo Scientific, USA). Expression of TLR3, TLR4, and TLR9 in DENV-2-infected and mock-infected cells was evaluated at 2, 8, and 24 hpi as we described in [25 (link)]. All data acquisition and analysis were done using the BD FACScan system and FACSDiva software, respectively.