Floid imaging station

The efficacy of FDN in instigating levels of ROS within NSCLC A549 cells was assessed through DCFH-DA stain as per the protocol described earlier [12 (link)]. For qualitative assessment, 2 × 10⁴ A549 cells/well were treated with 50, 100, and 200 µM of FDN and, at 12 h post-incubation, cells were stained by DCFH-DA (10 µM; 30 min at room temperature). Finally, the cells were visualized and the photomicrographs were captured through the green filter of FLoid Imaging Station (Thermo-Scientific, Waltham, MA, USA).
For quantification of ROS generated post-FDN treatment, 2 × 10⁴ A549 cells were seeded in a black-bottom 96-well plate and subsequently exposed to FDN (50, 100, and 200 µM) for 12 h under optimum culture conditions. Post-FDN exposure, cells were retreated with 10 µM DCFH-DA at room temperature for 30 min and recorded for their DCF-DA-instigated fluorescence intensity through Synergy H1 Hybrid Reader (BioTek, Winooski, VT, USA). The results were elucidated as percentage of mean DCF-DA intensity compared with the untreated control.

The cytotoxic effects of Far-C against HepG2 cells was estimated using MTT assay, described previously [30 (link)]. Approximately, 5 × 10³ HepG2 cells were allowed to adhere in each well of a 96-well plate under standard culture condition. Post-adherence, these cells were exposed to varying concentrations of Far-C (15, 30, and 60 µM) for 24 h. Subsequently, the media in each well was decanted and the wells were supplemented with 10 µL MTT (5 mg/mL) and the plate was incubated at 37 °C for another 4 h. Thereafter, 100 µL DMSO was further supplemented in each well and the plate was left for 30 min at 37 °C in darkness. Finally, the absorbance of solubilized formazan crystals was recorded at 570 nm (Bio-Rad spectrophotometer, Hercules, CA, USA). Far-C instigated cytotoxicity on HepG2 cells was expressed in terms of cellular viability percentage and was calculated as
$$\mathrm{Cellular}\mathrm{viability}\%=\frac{AbsorbanceofTreatedcells}{AbsrobanceofUntreatedcontrolcells}\times 100.$$
The morphology of HepG2 cells post-exposure with Far-C cells was visualized using microscope. The same procedure as mentioned above was repeated and HepG2 cells were exposed to 15, 30, and 60 µM Far-c and incubated for 24 h. Post incubation, the changes in the morphology of HepG2 cells were visualized under bright light of Floid imaging station (Thermo-Fischer Scientific, Waltham, MA, USA).

ROS-mediated oxidative stress in Far-C-treated HepG2 cells was evaluated using DCFH-DA stain through a microscope as reported previously [32 (link)] with subtle alterations. A total of 1 × 10⁵ HepG2 cells/well were allowed to adhere overnight in a 96-well plate. Post adherence, the cells were exposed to varying concentrations of Far-C (15, 30, and 60 µM) for 6 h. Subsequently, the media in each well was decanted and replaced with 20 µM DCFH-DA and the plate was further incubated in darkness for 30 min at 37 °C. Finally, the cells were visualized for their DCF-DA-mediated green fluorescence using Floid imaging station (Thermo-Fischer Scientific, Waltham, MA, USA).

Changes within nuclear morphology resulting in the onset of apoptosis of A549 cells post-exposure with GAN, MTX, and their combination were assessed through Hoechst 33,342 dye [47 (link)]. 1 × 10⁵ cells were exposed to the stated concentration of GAN and MTX either alone or in combination for 24 h. Subsequently, the cells were washed with PBS and exposed to 5 µg/mL of Hoechst 33,342 dye for 10 min in ambient culture conditions. Finally, the treated cells were visualized and recorded using blue filters with a FLoid Imaging Station, Thermo-Scientific. The level of characteristic blue fluorescence in different groups was qualitatively compared with that of the untreated control A549 cells.

Androgen-independent human-derived prostate cancer DU145 cells and J774A.1 murine alveolar macrophages were procured from the National Centre of Cell Sciences, Pune, India. The cells were allowed to proliferate in RPMI 1640 and DMEM high-glucose media supplemented with 10% FBS and 1% antibiotic–antimycotic solution, both v/v. The cells were continuously provided a humidified atmosphere with 5% CO₂ at 37°C. All the imaging reported in the present study was carried out using bright light and various fluorescent channels of the FLoid Imaging Station (Thermo Fisher Scientific, Waltham, Massachusetts, United States).

Briefly, A549 cells (5 × 10³ per well) were co-cultured with isoeffective doses (IC₂₅, IC₅₀, and IC₇₅) of MTX-ZnONPs, MTX alone, and ZnONPs alone for 24 h. Subsequently, the changes in cellular morphology of cells in all the treatment groups were examined, and images were taken using the relief phase channel of the FLoid imaging station (ThermoScientific, United States).