Synergy hybrid multi mode microplate reader

The intracellular ROS generation in HeLa cells was performed with the help of fluorescence microscopic imaging after the exposure of different concentrations of piperine at 12 h, using the fluorescence probes 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously reported (Ahamad et al., 2014[1 (link)]). The intracellular fluorescence of cells was observed using fluorescence inverted microscope (Nikon ECLIPSE Ti-S, Japan) and images were captured. Further, for the quantitative analysis of ROS, 1×10⁴ HeLa cells per well were seeded in 96-well black bottom cell culture plate and treated with 25 µM, 50 µM and 100 μM concentrations of piperine. After the treatment period, cells were incubated with DCFH-DA (10 mM) and fluorescence intensity of cells was observed by the multiwell microplate fluorimeter (Synergy Hybrid Multi-Mode Microplate Reader, BioTek) (excitation: 485 nm; emission: 528 nm). The percentage of fluorescence intensity was expressed as the relative fluorescence percentage of treated and control group.

Cells were plated at 9000 cells per cm2 and left overnight to attach. Cells were fixed with 4% paraformaldehyde in 1X PBS, pH 7.4 (warmed to 37 °C) for 20 min followed by distilled water wash. Samples were incubated with 0.1% crystal violet for 20 min at room temperature followed by 4 distilled water washes. Samples were left to air dry at room temperature for a minimum of 2 h followed by 10% acetic acid incubation for 20 min at room temperature, while shaking, to dissolve the crystal violet. Quantification was performed by measuring absorbance of samples at 590 nm relative to each other using Synergy Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Highland Park, VT, USA). Procedure was performed 3 times corresponding to the day of the experiment (days 1, 3, and 5) for each sample.

To investigate the ROS-mediated growth inhibitory effects of rutin NAC, a ROS inhibitor was used. As described in the previous sections, Caski cells were pre-treated with 10 mM NAC followed by rutin and stained with DCFH-DA for 30 min at 37 °C. Finally, fluorescence intensity was measured by using a multiwell microplate fluorimeter (Synergy Hybrid Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). To further examine the effect of ROS generation on cell growth inhibition in rutin-treated Caski cells, we performed an MTT assay in the presence of NAC (10 mM).