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.
Incucyte zoom version 2016b
Manufactured by Sartorius
The IncuCyte ZOOM version 2016B is a live-cell imaging system designed for real-time, automated analysis of cell behavior and morphology. It provides a controlled environment for cell culture and continuous monitoring of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Incucyte live cell analysis system, by Sartorius (3 mentions)
Cytotox green reagent, by Sartorius (2 mentions)
Incucyte caspase 3 7 green dye, by Sartorius (1 mentions)
Incucyte caspase 3 7 dye, by Sartorius (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Incucyte device, by Sartorius (1 mentions)
4 protocols using incucyte zoom version 2016b
1
Real-Time Spheroid Cytotoxicity Assay
2
Apoptosis Activation Quantification
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The IncuCyte Caspase 3/7 green dye (Sartorius) was used to determine the activation of essential mediators of apoptosis caspases 3 and 7. The IncuCyte Caspase 3/7 Dye is specially formulated for use in the IncuCyte Live-Cell Analysis System (Sartorius). Cells (3 × 103) were seeded in a 96-well plate containing complete DMEM the day before treatment. 30 min before adding pPBS, 5 μM Caspase 3/7 green dye was added to each well. In addition, 1 h prior to treatment, 1 mM of N-acetyl cysteine (NAC) was added to some cells. After treatment the plate was incubated in the IncuCyte and images were taken every 2 h over a period of 24 h. Data were analyzed with the IncuCyte ZOOM version 2016B (Essen BioScience) to collect the total green object count per well, defined as the number of apoptotic cells per well.
3
Quantifying Spheroid Cytotoxicity
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The Cytotox Green reagent (10 nM, Essen Biosciences) was used for real-time quantification of cell death. Plates were incubated in the IncuCyte Live-Cell Analysis System (Sartorius, Ann Arbor, MI, USA) and images were collected every 24 h for 7 days. Data were analysed with the IncuCyte ZOOM version 2016B (Essen BioScience) to collect the following metrics: (1) spheroid core area, corresponding to the proliferative region only (red live cells; calculated as the mean fluorescent object area); (2) total spheroid area, corresponding to the combined viable, proliferative 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) normalized to the untreated control at each time point, representing the cytotoxic effect of the treatment.
4
Intracellular ROS Quantification
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To determine the intracellular ROS, CellROX Green Reagent (Invitrogen) was used. The cell-permeant dye is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation and subsequent binding to DNA. Cells (3 × 103) were seeded in a 96-well plate containing complete DMEM the day before treatment. 30 min before adding pPBS, 2.5 μM CellROX Green Reagent was added to each well. In addition, 1 h prior to treatment, 1 mM of NAC was added to some cells. After treatment the plate was incubated in an IncuCyte device and images were taken every 2 h over a period of 24 h. Data were analyzed with the IncuCyte ZOOM version 2016B (Essen BioScience) to collect the average green object mean intensity of each well.
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