Moticam 2500 camera

For the histological analysis, pieces of liver from each animal were fixed in 10% formalin (Ref Sigma Aldrich, St. Louis, MI, USA) and embedded in paraffin. Next, 4 µm-thick sections were cut and stained with hematoxylin and eosin (H&E). The images were acquired in a Digital Upright Microscope BA310 Digital and a Moticam 2500 camera. The selection of test objects was performed according to color and choosing the same limits for the binarization of all images. At least three pictures from different regions of each cut were taken.

The 3D RMS spheroids were grown on 1% (w/v) agarose-coated 96-well plates. To prepare the plate, powder-form low electroendoosmotic (EEO) agarose (Sigma-Aldrich, Poole, UK) was fully dissolved in 1 × PBS with very mild heating until the formation of a clear solution (Sigma- Aldrich, Poole, UK). Warm agarose solution (100 μL) was carefully added a the flat-bottom 96-well plate, ensuring that no bubbles were present. The plate was left to dry and sterilized under UV for 2 h at room temperature. A flat solid agarose layer could be seen to form in the well after this step. RMS cells from cultures were washed with DMEM medium and seeded at a density of 100,000 cells per well onto the agarose-coated plate without damaging the agarose layer. The plate was then cultured at 37 °C with 5% CO₂ in a tissue culture incubator for 4 days. The growth of the spheroids was monitored every day, and after 4 days they were transferred into a normal 96-well plate for experiments.
The RMS spheroids were incubated for 72 h with either (i) PBS, (ii) the same concentration of free IR820 NIR dye, or (iii) IR820 loaded siRNA linked MSNPs. The sizes of the spheroids were monitored at t = 0 and every 24 h afterwards using a Motic 101M microscope with a Moticam 2500 camera and MHG 100B laser (Motic, Xiamen, China). The diameters of spheroids were quantified using ImageJ (NIH, Bethesda, USA).

The MTT reduction assay [28 (link)] was carried out as an indicator of cell viability and mitochondrial function. After treatments, medium was removed and cell viability was measured by incubating cells with 100 μL of MTT (2 mg/mL) for 1 h and at 37°C. Formazan blue crystals generated by living cells were dissolved in DMSO and the absorbance was measured at 550 nm using a Spectrostar Nanomicroplate reader (BMG Labtech, Ortenberg, Germany). Results are expressed as the percentage of viable cells in comparison to control cells (taken as 100% absorbance). MTT assay was used for assessing the effects on cell viability of several concentrations of ginsenosides (by themselves), as well as for the evaluation of their cytoprotection against rotenone (pre-treatments with ginsenosides and later addition of rotenone).
Cellular morphology was examined after the different treatments by phase contrast microscopy and photographs were taken using a Motic Moticam 2500 camera.