Sybr 14 dye

Oxidative stress was detected by using CellROX deep red reagent and MitoSOX red mitochondrial superoxide indicator (both from Molecular Probes, Eugene, MN, USA). CellROX exhibits fluorescence (emission maxima at 650 nm) upon oxidation by reactive oxygen species, and MitoSOX red (emission maxima at 580 nm) detects superoxide in the mitochondria of live cells. Spermatocytes were treated with CSC or DMSO for 1 h and 5 h as previously reported.^{13 (link)} Then, CellROX and MitoSOX reagents were added to a final concentration of 1 μM and 5 μM, for 30 and 10 min, respectively, at 37 °C. The cells were washed with warm buffer as per the manufacturer’s protocol, counter-stained with SYBR 14 dye or TO-PRO-3 (Molecular Probes; 1 : 1,000) for 5 min at room temperature, and then subjected to flow cytometry.

C33-A cells were seeded onto glass chamber slides (Lab-Tek, Nalgene Nunc, Rochester, NY, USA) and infected as mentioned above. Infected or non-infected controls were washed with phosphate-buffered saline (PBS) and fixed in ethanol/acetone 50 % v/v for 10 min at room temperature. Cells were permeabilized with 0.2 % Triton-X100 for 2 min in order to detect the viral pr-M protein inside infected cells and incubated in blocking solution (PBS with 10 % FBS and 0.2 % Tween 20). Cells were labelled with anti-dengue virus pr-M antibody (Abcam, ab41473) or anti-IL28R (Santa Cruz Biotechnology, sc-66541), washed and incubated with rabbit anti-mouse IgG-Texas red (Abcam, ab6726) or bovine anti-goat IgG-CFL-647 (Santa Cruz Biotechnology, sc-362284) for 1 h at room temperature. SYBR-14 dye (Molecular Probes, Eugene, OR, USA) was used to stain the nuclei. The stained cells were visualized by laser confocal microscopy system E600-C1 using the image analysis software EZ-C1 (Nikon).