4.2. Viral Quantification and Immunofluorescence Assay (IFA)

Virus quantification began with ten-fold serial dilution of viral supernatant. The dilute solution was added to Vero cells for 1 h absorption and then replaced with 1.5% methylcellulose for 2-day incubation. Viral titers were calculated as fluorescent focus units (FFU) per ml. TCID₅₀ assays were performed by adding the above-mentioned dilutions to Vero cells in 96-well plates. Each dilution was repeated 6 times. Following incubation for 4–5 days, DENV-induced cytopathic effect was observed under microscope.

IFA involved incubating virus cells with the primary antibody and then the secondary antibody at room temperature following fixation. PBS washing was performed three times between each step. Images were captured using an inverted OLYMPUS IX73 fluorescence microscope (Olympus, Tokyo, Japan). Anti-DENV NS3 protein antibodies (Catalog number: GTX124252; 1:400 dilution; GeneTex, Irvine, CA, USA) and anti-rabbit DL488 (Catalog number: GTX213110-04; 1:1000 dilution; GeneTex, Irvine, CA, USA) were used for IFA. Anti-Flaviviridae envelope antibodies (Homemade; 1:400 dilution) and anti-mouse DyLight488 (Catalog number: GTX213111-04; 1:1000 dilution; GeneTex, Irvine, CA, USA) were used for FFU. DAPI was used to stain the cell nucleus.