Triton x 100 0.5

From 8 × 10⁵ to 1.6 × 10⁶ hBM-MNCs were seeded in 2-well culture chamber slides (Thermo Scientific) and cultured in DMEM/10%PhABS for 6 days, as described above. Cultures were then washed twice in D-PBS and MPCs were fixed in paraformaldehyde 4% for 15′. Permeabilization was performed by incubation in Triton X-100 0.5% (Sigma Aldrich) for 15′, after the removal of the fixative by extensive wash in D-PBS. Slides were blocked applying Image-iT™ FX signal enhancer (Thermo Scientific) and incubated with anti- human nestin monoclonal antibody (Abcam, Cambridge, UK) overnight. Antibody excess was removed by washing in Triton X-100 0.5% and slides were then incubated with AlexaFluor® 488-conjugated secondary antibody for 1 h. F-actin staining was performed by AlexaFluor® 555-conjugated phalloidin (Thermo Scientific) for 20′. The slides were finally mounted with ProLong® anti-fade reagent with DAPI and pictures were taken using an inverted fluorescence DM IRB microscope (Leica, Wetzlar, Germany) equipped with LAS AF image analysis software (Leica).

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.

The hemolytic activity was estimated according to the method described by Muhammad et al. (2016) [47 (link)]. Triton X-100 (0.5%) (Sigma Aldrich, St Louis, MO, USA) was used as a positive control. The absorbance was measured using a UV-2800 BMS Scientific Technical Corporation (PVT) Ltd. Spectrophotometer (Shanghai, China) at 550 nm.