Cellomics multiparameter cytotoxicity 3 kit

Cellomics multiparameter cytotoxicity 3 kit (Thermo Scientific) was used as described previously [21 ]. 1 × 10⁴ cells per well were plated in a 96-well plate and incubated overnight at 37°C in 5% CO₂. The cells were treated with different concentrations of the PDM and further incubated at 37°C in 5% CO₂ for 24 hours. MMP dye and the cell permeability dye were added to live cells and incubated for 1 hour. After fixing the cells, the nucleus was stained with Hoechst 33258. Stained cells were visualized and images were captured using Cellomics ArrayScan HCS reader (Thermo Scientific).

Cellomics multiparameter cytotoxicity 3 kit (Thermo Scientific™, Pittsburgh, PA, USA) was used to investigate the vital apoptotic events in HCT-116 cells in the presence of Dis. Cytotoxicity 3 assay was used to provide information about the changes in nuclear morphology, cell membrane integrity, cytochrome C release and mitochondrial outer membrane permeabilization (MOMP). HCT-116 cells were seeded in 24 well-plate and covered by a cover slide followed by treatment with Dis for 24 h, 48 h and 72 h. Treated cells were stained with YoYo dye (life technology, CA, USA) and Mitotracker dye (life technology, CA, USA) followed by fixation and blocking procedures according to the manufacture’s protocol. Primary cytochrome C (Thermo Scientific™, Pittsburgh, PA, USA) antibody and secondary DyLight 650 (Thermo Scientific™, Pittsburgh, PA, USA) were added to each well and incubated for 1 h in the dark. Hoechst 33342 dye (Thermo Scientific™, Pittsburgh, PA, USA) was added in the last step to stain the nuclei of the cells. Cells were washed and cover slides were transferred on the slide and introduced to confocal Leica TCS SP5 II microscope (Leica Microsystems, Mannheim, Germany). Intensity of each dye was measured by using LAS X software.

Multiple cytotoxicity assays were conducted using the Cellomics^® Multiparameter Cytotoxicity 3 Kit (Thermo Scientific, PA, USA). A total of 5 × 10³ cells were seeded in each well of 96-well microplate. The cells were then treated with pulchrin A at three concentrations (11, 22 and 33 μM) for 24 h and then incubated overnight at 37°C. After incubation, several solutions were continuously added in each well containing 50 μL of live cell staining solution, 100 μL of fixation buffer, 100 μL of 1 X permeabilization buffer and 100 μL of 1 X blocking buffer, which were incubated for 20 min, 10 min, 30 min and 15 min, respectively. Two antibody solutions (cytochrome c primary antibody and DyLight^™ 649 Conjugated Goat Anti-Mouse IgG secondary antibodies) were added at the final stage of assay preparation. The plate was then read and evaluated on the ArrayScan, high content screening (HCS) Reader from Thermo Fisher Scientific (Pittsburgh, PA, USA).

Multiple cytotoxicity assays were conducted using the Cellomics^® Multiparameter Cytotoxicity 3 Kit (Thermo Scientific, Pittsburgh, PA, USA) as mentioned in details by Cheah et al. (2011) (link). HeLa cells were analyzed using the Array Scan HCS system after 24, 48, and 72 h of treatment with IC₅₀ concentration of AM (24 µM).