2.3. Dengue Virus Titration

Focus-forming unit assay (FFU) was performed for virus titration. Vero cells were cultivated in 96-well plates at 3 × 10⁴ cells per well for 1 day or until confluence. Monolayer cells were infected with a serial 10-fold dilution of viral supernatants for 2 h in a 37 °C, 5% CO₂ incubator. Virus supernatant was added with 100 μL of overlay medium (2× MEM medium supplemented with 3% FBS and 1.5% carboxy methyl cellulose) for 3 days in a 37 °C, 5% CO₂ incubator. Infected cells were then stained with antibody against viral protein and the viral titer was counted by FFU assay. Briefly, infected cells were washed and fixed with 3.8% formaldehyde. Cells were then permeabilized with 1% Triton X-100 and stained with a 4G2 antibody (anti-E DI/II). Finally, secondary antibody labeled with horse radish peroxidase (HRP) was added to react with mouse Igs, followed by color detection with substrate for HRP to observe the focus-forming units of infected cells. Stained foci were counted under an inverted light microscope (Olympus CKX41; Olympus Corporation, Tokyo, Japan) at a magnification of 100×, and that value was used to calculate the viral titer, which was reported as focus-forming units (FFU)/mL.