Cytotox green reagent

For cytotoxicity death assay, cells were seeded in 96-well plates in low density (5,000 cells/well) and incubated overnight. 1000X Cytotox Green Reagent or Caspase 3/7 Green Apoptosis Assay Reagent (Essen Bioscience) was diluted in medium and working dilutions of the drug were prepared in Cytotox Green or Caspase3/7 Green supplemented media. After treatment, plates were loaded in IncuCyte Zoom and images were acquired in real-time for phase to quantify growth. Activity of green reagent was simultaneously acquired at the green channel to quantify death. IncuCyte Zoom software was used for the analysis and data export.

Pemetrexed was purchased from Sigma. For in vitro treatment cells were plated in a 96-well plate (4000 cells/well) and incubated overnight. For cytotoxicity death assay, 2000X Cytotox Green Reagent (Essen Bioscience) was diluted in RPMI and working dilutions of Pemetrexed was prepared in Cytotox Green supplemented media. After treatment, plate was loaded in Incycuyte Zoom and images were acquired in real-time for phase to quantify growth. Activity of Cytotox reagent was simultaneously acquired at the green channel to quantify death. Incycuyte Zoom software was used for the analysis and data export.

Pemetrexed was purchased from Sigma, dihydrothymine (DHT) was purchased from Selleckchem and were dissolved in DMSO to final concentration of 100 mM and 40 mM respectively. Cisplatin was purchased from Santa Cruz Biotechnology and was dissolved in PBS to the final concentration of 80 mM. For in vitro treatment cells were plated in 96-well plates (MDA-MB-231 = 3000–4000 cells/well and T-47D = 6000 cells/well) and incubated overnight. For cytotoxicity death assay, 2000X Cytotox Green Reagent (Essen BioScience) was diluted in RPMI and working dilutions of Pemetrexed and Cisplatin were prepared in Cytotox Green supplemented media. Working dilutions of DHT were prepared in RPMI (without Cytotox Green). After treatment, plate was loaded in Incycuyte Zoom and images were acquired in real-time for phase to quantify growth. Activity of Cytotox reagent was simultaneously acquired at the green channel to quantify death. Incycuyte Zoom software was used for the analysis and data export.

We used the Cytotox Green reagent (50 nM, Essen Biosciences) in media for real-time quantification of cell death. This cell-permeable compound dye binds to nuclear and mitochondrial DNA and becomes strongly fluorescent upon oxidation. The plates were incubated in the IncuCyte Live-Cell Analysis System (Sartorius, Ann Arbor, Michigan, MI, USA) and spheroid growth was followed during seven days after treatment. Data were analyzed with the IncuCyte ZOOM version 2016B (Essen BioScience) as well as ImageJ software to collect the following metrics: (1) spheroid core area, corresponding to the proliferative region only (red live cells; calculated by drawing a circle at the edge of the bright red core of the spheroid using ImageJ); (2) total spheroid area, corresponding to the combined viable, proliferative spheroid core and the Cytotox Green⁺ region (total spheroid region measured from phase contrast images); and (3) the amount of Cytotox Green⁺ in treated spheroids (confluence percentage of the image area occupied by green objects using ImageJ software) normalized to the untreated control at each time point, representing the cytotoxic effect of the treatment.

Cells were plated in 96-well plates (Olympus Plastics) at 5000 cells/well. After 24 h, growth medium was replaced with fresh medium containing compounds of interest and Cytotox Green Reagent (Essen BioScience, Ann Arbor, MI, USA). Dead cells were detected in real time for 72 h using the IncuCyte S3 cell imaging system (Essen BioScience). The relative cytotoxicity level was estimated as the number of green fluorescent objects normalized to corresponding cell confluence values. Staurosporine (200 nM) was used as a positive control to induce cell death.