**3.6. Evaluation of mitochondrial membrane potential using JC-1**

To evaluate mitochondrial membrane potential, the dye JC-1 (5, 5′, 6, 6′-Tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide) was used (Thermofisher.com). In healthy mitochondria with high membrane potential, JC-1 forms red fluorescent aggregates. In contrast, JC-1 occurs as green fluorescent monomers in mitochondria with depolarized membrane potential. Semiconfluent GBM cell layers seeded into 24-well microtiter plates (Corning.com) (density: 17,000 cells per cm2 ) were stained one day after culture with 2 μg/ml JC-1 for 30 min. After incubation, the culture medium was removed and cells were washed twice with pre-warmed HBSS. After washing, the fresh culture medium was added and cells were treated for 4 h with 0.01% DMSO (vehicle control) or 7 μM Vac. Imaging was performed by IncuCyteZOOM® and red/green total fluorescence per image was quantified and collected using IncuCyteZOOM® software. The ratio between red and green fluorescence was calculated. A shift from red to green fluorescence and therefore, a decline in the red/green ratio indicates mitochondrial depolarization.

**Figure 4A** (https://www.intechopen.com/books/autophagy-in-current-trends-in-cellular-physiology-and-pathology/introductory-chapter-overview-on-autophagy-in-burden-of-functions).

Mitophagy-Related Cell Death Mediated by Vacquinol-1 and TRPM7 Blockade in Glioblastoma IV

EMS drafted the manuscript (ms), PW performed STEM microscopy, 3D tomography and reconstruction, PS performed experiments, designed figures and contributed to the ms text; BM performed experiments and drafted the ms, MH, and PSch contributed to chemical property analysis of Vac, HM contributed to trans electron microscopy, AP, CRW, and

, Michael Hinz4

, Michael Georgieff<sup>1</sup>

, Haouraa Mostafa1

http://dx.doi.org/10.5772/intechopen.77076

91

and

,

MG edited the ms. All authors reviewed and approved the final version of the ms.

, Barbara Moepps3

, C. Rainer Wirtz<sup>6</sup>

1 Department of Anesthesiology, University Hospital Ulm, Ulm, Germany

2 Central Facility for Electron Microscopy, University of Ulm, Ulm, Germany

3 Institute of Pharmacology and Toxicology, University Hospital Ulm, Ulm, Germany

5 Center for Mitochondrial and Epigenomic Medicine, Children's University Hospital of

[1] Jendrossek V, Belka C, Bamberg M. Novel chemotherapeutic agents for the treatment of glioblastoma multiforme. Expert Opinion on Investigational Drugs. 2003;**12**:1899-1924.

[2] McGranahan N, Furness AJS, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, Jamal-Hanjani M, Wilson GA, Birkbak NJ, Hiley CT, Watkins TBK, Shafi S, Murugaesu N, et al.

6 Department of Neurosurgery, Bezirkskrankenhaus Günzburg, Günzburg, Germany

\*Address all correspondence to: marion.schneider@uni-ulm.de

Philadelphia Research Institute, Philadelphia, PA, USA

DOI: 10.1517/13543784.12.12.1899

**Conflict of interest**

**Authors contributions**

**Author details**

Philip Sander1

**References**

Patrick Schaefer<sup>5</sup>

E. Marion Schneider1

The authors declare no conflicts of interest.

, Paul Walther2

\*

4 M. Hinz UG, Neuried, Germany

, Andrej Pala<sup>6</sup>
