**3.5. Detection of ROS**

the massively upregulated mitophagy in 4 h Vac-treated GBM does not appear to occur with microtubule-assisted mitochondrial fission, as demonstrated in stressed tumor cells [18] since microtubules appear to be randomly distributed (https://mts.intechopen.com/

**Figure 4.** Ultrastructural changes induced by Vac in GBM cells (#12537-GB). Virtual sections of #12537-GB cells investigated by STEM tomography demonstrate active mitophagy in control cells with early phagophore formation around mitochondria (white stars) by smooth ER cisternae (black arrows); in addition, numerous autophagolysosomes (black arrowheads) are shown with fully digested cytoplasmic as well as organelle derived material (A), this video section is part of the video tomogram provided as supplementary file 1 [16]; following Vac-treatment (4 h, 7 μM), phagophore formation is not prominent, ER appears to be swollen (black arrow as an example), and autophagolysosomes (white arrowheads) are more abundant. The material ingested is not fully digested, residual mitochondria can be identified by their morphological remnants (white arrows) (B). https://mts.intechopen.com/download/index/process/155/authkey/5f

The glioma cell line #12537-GB has been previously described [6]. The #12794-GB cell line has been established from a female patient's tumor material received from the neurosurgical department of the Bezirkskrankenhaus in Günzburg (Universal trial number: U111-1179-3127) with patient-informed consent. The tumor material was minced and cells from the tumor material were taken into the culture by trypsinization of the tumor material (2.5% trypsin), followed by Ficoll separation. Continuous cultures were performed in Iscove's Modified Dulbecco's Medium (IMDM) (Lonza.com, USA) supplemented with 10% fetal calf serum (FCS, endotoxinfree, Batch 0247x, Merck/Biochrom.com, Germany), GlutaMAX (ThermoFisher.com, USA), and

cultured in IMDM containing 10% endotoxin-free FCS (Batch No: 0247x, Merck/Biochrom.com).

. Hs68 fibroblasts (Hs68-Fi) were purchased from ATCC, and

download/index/process/155/authkey/5fc95d7040949214f944031367b18403).

**3. Materials and methods**

c95d7040949214f944031367b18403.

88 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**3.1. Cell lines and cell culture**

antibiotics at 37°C under 5% CO2

To detect ROS, carboxy-H2 DCFDA (CM-H<sup>2</sup> DCFDA) (Thermofisher.com, USA) was used. In the presence of ROS, the dye gets oxidized and emits green fluorescence. GBM cells were seeded into flat-bottom 96-well microtiter plates (Sarstedt.com). One day after seeding, semi-confluent GBM layers were treated with 0.01% DMSO (vehicle control) or 7 μM Vac. After treatment, CM-H2 DCFDA (stock 5 mM in DMSO) was added in a final concentration of 5 μM to the culture medium (IMDM, 10%FCS). Imaging was performed by IncuCyteZOOM®. Total green fluorescence per image was quantified and collected by IncuCyteZOOM® software.
