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Application Notes

The Response of Mitochondria in Cancer Spheroids to Drugs

The effect of an uncoupling agent on mitochondria in spheroids can be visualized.
This is a promising method for testing toxicity to mitochondria.


Mitochondria are indispensable organelles that supply cellular energy. Toxicity to mitochondrial function is an important factor to be tested for new compounds. In this study, the response of mitochondria in 3D cell cultures to drugs was investigated.


Preparation of samples

A cell suspension of HT-29 was seeded to a U-bottom 96-well plate and  incubated for 96 hours to prepare 3D cell cultures. Spheroids were incubated with MitoTracker® Red CMXRos (a membrane potential-dependent, mitochondrial staining dye) and FCCP (uncoupling agent, 0-300 μM) for 45 minutes, followed by fixing in 4% formalin and  nuclear staining with DAPI.

Acquisition and analysis of images

Confocal fluorescent images of the above-mentioned spheroids were obtained. The average luminosity of nuclei and mitochondria in a cell was estimated by superimposing images obtained every 5 μM using the mean intensity projection method. No effect of FCCP was observed on nuclei (Figure 1 (a)), while the average luminosity of mitochondria in a cell decreased with an increase in FCCP concentration (Figures 1 (b) and 2).


MitoTracker is a registered trademark of Molecular Probes, Inc.
Olympus is a registered trademark, and NoviSight and Insightful Analysis, Intelligent Answers are trademarks of Olympus Corporation.

Products related to this application

3D Cell Analysis Software


NoviSight 3D cell analysis software provides statistical data for spheroids and 3D objects in microplate-based experiments. Use it to quantify cell activity in 3D, easily capture rare cell events, obtain accurate cell counts, and improve detection sensitivity. NoviSight software works with a range of imaging techniques, including point-scan confocal imaging, two-photon imaging, spinning disk confocal imaging, and super resolution live cell imaging.

  • Fast 3D image recognition from whole structures to subcellular features
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  • Equipped with a variety of ready-to-use default assays or easily design your own

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