Dengue virus titer determination

Immunofluorescence staining was used to visualize infected cells [47]. Briefly, twofold serial dilutions of dengue virus type 2 were added in triplicate to 20,000 Vero cells plated day before in DMEM with 2 % fetal bovine serum, 100 U of penicillin/ml and 100 μg of streptomycin/ml. After 72 h of incubation at 37 °C in 5 % CO₂, cells were fixed with 4 % paraformaldehyde and permeabilized with 0.2 % Triton X-100. Then, cells were washed with 1X PBS and incubated overnight at 4 °C with dengue virus type 2 serotype-specific mouse monoclonal antibody, which was harvested from HB-46 cells. Wells were washed three times with 1X PBS, incubated 90 min with Cy3-labeled donkey anti mouse IgG (Jackson Immunoresearch Europe) and documented using fluorescence microscope Olympus IX-81 with camera (Hamburg, Germany). ImageJ software (NIH) was used for image analysis and evaluation of positive and negative wells.