Eclipse ts100 inverted routine microscope

Live and dead cells were evaluated by AO/EB (Acridine orange and ethidium bromide) fluorescent staining (Hameed et al., 2016 (link)). Overnight grown E. coli cells (10⁷ cells/ml) were treated with or without carvacrol (MIC) for 15 min at 37°C. After incubation, cells were harvested by centrifugation, washed with PBS and stained with AO/EB (1:1) (100 μg/ml) for 30 min. After 30 min incubation, cells were washed with PBS and visualized under fluorescent microscope. Similarly, to assess the effect of carvacrol on reactive oxygen species (ROS) and membrane potential, H₂DCFDA [5(6) -Carboxy-2′,7′-dichlorofluorescein diacetate] and rhodamine 123 fluorescent staining was carried out, respectively (Warnes et al., 2012 (link)). In brief, E. coli cells (10⁷ cells/ml) were treated with or without carvacrol for 15 min followed by centrifugation to harvest the cells. After washing with PBS, cells were stained in dark for 30 min with H₂DCFDA (10 mM) or rhodamine 123 (1 μg/ml). Cells were thoroughly washed with PBS and visualized under EPI fluorescent Eclipse TS100 Inverted Routine Microscope with PAXCam software (Nikon TS 100, Japan). All experiments were repeated three times independently and at least three different fields were observed for each culture.

C6/36 (2 × 10⁵) cells were seeded into 24-well plates before infection with DENV2 and ZIKV (M.O.I. of 1) as described^{42 (link)}. Cardol triene at 10 µM were introduced to the DENV2 and ZIKV infected cells during and after infection. DMSO was used as a 0% inhibition control and mouse anti-Flavivirus envelope protein antibody (4G2) was used as a 100% inhibition control. Cells were incubate at 28 °C for 2 days before addition of 0.5 M 2-(N-morpholino) ethanesulfonic acid (MES) (pH 5.0) (Sigma Aldrich, St Louis, USA). Moreover, cells from selected wells subsequently underwent DAPI staining. Cell-cell fusion was observed under brightfield and fluorescence using an Eclipse TS100 Inverted Routine Microscope (Nikon, New York, USA).