Ds ri1 12.7 megapixel camera

For the determination of live, apoptotic and necrotic cells, assay was performed as described by Ribble et al. [56] (link) Cocktail of EB and AO (100 μg/mL) was prepared in phosphate-buffered saline. This assay is based on apoptosis induced characteristic nuclear condensation and fragmentation, whereas necrosis is characterized by the inability to exclude vital dye, leading to orange staining of nuclei. This procedure was used for qualitative analysis of apoptotic and necrotic cells after the treatment of (1.4 μg/mL) of free Cur; (5.5 μg/mL) of free Quer; (1.6 μg/mL) of free Asp; (5 and 10 μg/mL) of CS-SHMP-CQA-NPs and control, cells were incubated for 30 min with cocktail of EB/AO (100 mg/mL). Free drug treatments are chosen as per the drug content in the highest dose of CS-SHMP-CQA-NPs i.e. 10 μg/mL. The apoptosis/necrosis was observed by fluorescence images in upright microscope (Nikon Eclipse 80i equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan). For each group, all cells in four image frames were analyzed using NIH ImageJ analysis software (USA) by following literature reports [57] , [58] . Green stained cells were counted as live cells and bright orange/red stained cells were counted as apoptotic/necrotic cells. Percent (%) apoptotic cells were measured for each group [57] .

Qualitative and quantitative change of MMP was measured by JC-1 stain (Sigma-Aldrich kit). In healthy cells, JC-1 forms J-aggregates which display strong fluorescent (red) intensity with excitation and emission at 560 and 595 nm, respectively. However, in apoptotic cells, JC-1 exists as monomers which show strong fluorescent (green) intensity with excitation and emission at 485 and 535 nm, respectively. Cells were exposed to (1.4 μg/mL) of free Cur; (5.5 μg/mL) of free Quer; (1.6 μg/mL) of free Asp; (5 and 10 μg/mL) of CS-SHMP-CQA-NPs for 24 h. Free drugs treatments are chosen as per the drug content in the highest dose of CS-SHMP-CQA-NPs i.e. 10 μg/mL. After treatment, cells were incubated with 5 μM JC-1 (ENZO life sciences) stain for 30 min at 37 °C, washed and images were visualized by fluorescence microscope [59] (link) (Nikon Eclipse 80i equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan). Fluorescent intensity per cell in four image frames for each group was quantified using NIH ImageJ analysis software (USA) by following reports [60] (link), [61] (link), [62] (link), [63] (link). Mitochondrial depolarization in each group was quantified by calculating the ratio of red to green fluorescent intensity and was normalized to the control (red-to-green fluorescent ratio of control considered as 1) [62] (link), [63] (link), [64] (link).

The SKOV-3 cell death induced by MTX was determined by AO and EtBr staining followed by Kitazumi et al protocol.18 (link) SKOV-3 cells were exposed to MTX (15, 25, and 50 µM) for 24 hours. After incubation, the treated cells were trypsinized and centrifuged at 1,500 rpm for 5 minutes. The cells were then re-suspended in cold PBS and again centrifuged at 1,500 rpm for 5 minutes to obtain fresh clean pellets. The pellets were mixed with fluorescent dye staining solution, that is, cocktail of AO/EtBr (1:1). The freshly stained cell suspension was dropped onto a glass slide and observed under a fluorescence microscope (Nikon Eclipse 80i with Nikon DS-Ri1 12.7 megapixel camera). The morphological criteria used for the classification of healthy, apoptotic, and necrotic cells are according to the recommendations from the INHAND Apoptosis/Necrosis Working Group.19 (link)