Ap conjugated anti mouse

DENV3-290 and DENV3-5532 titers were determined by focus formation assay, modified from the protocol described in Desprès et al. (1993) (link). C6/36 cells (Aedes albopictus) were kept in Leibovitz’s-15 media (L-15) supplemented with 5% FBS, 0.26% tryptose and 25 μg/ml gentamicin and plated 1 day prior to the assay. Serial dilutions of the DENV strains were used to infect C6/36 for 60 min at 28°C in L-15 media without FBS. After inoculum removal, the cells were incubated with supplemented L-15 media containing 1.6% CMC (Sigma-Aldrich) in a static incubator at 28°C for 7 days. The media was then removed, the cells were washed three times with 1× PBS, fixed with 3% paraformaldehyde (Sigma-Aldrich) for 20 min, and washed again three times with 1× PBS. Permeabilization was performed with Triton X-100 0.5% (Sigma-Aldrich, St. Louis, MO, United States) for 4 min, followed by washing with 1× PBS (3×) and incubation with 4G2 (1:100) for 45 min at 37°C. After washing, the cells were incubated with AP-conjugated anti-mouse (Promega, Madison, WI, United States) at 1:7,500 for 30 min at 37°C. After additional washing (3× with 1× PBS) the reaction was developed using AP buffer containing 6.6% NBT and 3.3% BCIP. The average for each dilution was calculated between the duplicates, and the amount of focus formation units per ml (FFU_C6/36/ml) was calculated.