After YH25448 research buy thawing at room temperature, the stock was used as inoculum for monolayers of naïve C6/36 cells in Leibovitz’s (L-15) medium containing 1% heat-inactivated fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB) and 1.2% antibiotic (Penicillin G and Streptomycin). At days 5-7 after challenge, the supernatant solution was removed and used as inoculum for subsequent trials. Naïve

C6/36 cells challenged with Dengue virus Culture plates (6-well, Costar, Corning) were seeded with C6/36 cells at a density 106 cells/well and incubated for 24 h at 28°C to produce confluent monolayers. The cell monolayers were then challenged with DEN-2 at a multiplicity of infection (MOI) of 0.1. After incubation for 2 h with gentle shaking at room temperature, the medium was removed

and fresh medium containing 2% FBS was added for further incubation at 28°C. Persistent infection of C6/36 cells with Dengue virus Persistent infections of DEN-2 in C6/36 cells were achieved as previously described [6]. Briefly, after 2 days incubation post DEN-2 challenge (acute infections in C6/36 cells), the supernatant solution was removed and cells were suspended by knocking in L-15 containing 10% FBS at 1:3 dilution and transferred to a new culture well at 1/2 density for 2-days cultivation Momelotinib datasheet (to full confluence) before repeating the decantation, suspension, dilution and transfer process sequentially at 2 day intervals to establish persistently infected cultures. Three replicates were done in 6 well plates at 2 day intervals. Mock-infected cells were run in parallel to the viral infected cells to serve as negative controls. Preparation of cell and virus free culture filtrates Culture supernatant solutions (4 ml) from cultures acutely infected or persistently infected with DEN-2 were clarified by learn more centrifugation at 2000 × g for 5 min. The supernatant was transferred to an Amicon Ultra filter unit (buy GDC-0941 Millipore) containing a cellulose,

low-binding membrane with a molecular weight cut-off of 5 kDa. The ultrafiltration device was centrifuged at 4000 × g for 25 min to produce a filtrate that consisted of substances that could pass the 5 kDa molecular weight cut-off. These filtrates were collected and stored at -20°C. Immunofluorescent staining for confocal microscopy For DEN-2 detection, cells were fixed with 4% formaldehyde in PBS for 15 min, washed twice with PBS, permeabilized with 0.1% Triton X-100 for 5 min and blocked with PBS containing 10% FBS. Cells were incubated for 1 hour with 3H5 monoclonal antibody against DEN-2 virus envelope protein followed by incubation for 30 min. with 1:500 dilution of fluorophore-labeled secondary antibody conjugate (Alexa Flour 488 goat anti-mouse IgG, A-11001, from Molecular Probes) directed against the primary antibody.