Mtt solution

To assess the treated cells’ metabolic rate as a sign of cell viability, we performed a 3(4,5 dimethylthiazol)-2,5 diphenyltetrazolium (MTT) assay as described by Sehm et al.^{31 (link)} After 24 h, incubation with either 5 or 10 μM sorafenib or erastin, cells were incubated with freshly made MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) for 4 h at 37 °C, 5% CO₂. We used 100 μl isopropanol, supplemented with 0.1 N HCl for the following cell lysis. The optical density of each well was determined using the microplate reader Tecan Infinite F50 (Crailsheim, Germany) set to 550 nm (wavelength correction set to 690 nm).

The cell viability assay was performed using 3(4,5 dimethylthiazol)-2,5 diphenyltetrazolium (MTT) assay according to Hatipoglu et al., 2015 [30 (link)] the cell viability was measured. 3.000 cells/well were plated in 96 well-plates one hours prior to the drug treatment. In case of the primary rat astrocytes culture, 4.000 cells/well were plated in 96 well-plates and the drug treatment were done after 4 days. On the fourth day after treatment cells were incubated with MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) for 4 h at 37°C, 5% CO₂. For the ferroptosis measurement 20.000cells/96well were seeded, drug treatment occurred 1 h after seeding and the MTT solution was added after 24 h of incubation. The lysis of the cells occurred with 100 μl isopropanol + 0.1 N HCl. The optical density of each well was determined using the microplate reader Tecan Infinite F50 (Crailsheim, Germany) set to 550 nm (wavelength correction set to 690 nm) using Magellan software. Plates were normally read within 1 h of adding after lysis. Control was cells without drugs. The viability of the cells was expressed as the percentage of control. Assays were performed on at least three independent experiments.

The cell viability assay was performed using 3(4,5 dimethylthiazol) - 2,5 diphenyltetra-zolium (MTT) assay according to Hatipoglu et al. [36 (link)]. 3000 cells/ well were plated in 96-well plates, the drug treatment followed two hours later. After 72 hours cells were incubated with MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) for 4 h at 37°C, 5% CO₂. The following lysis of the cells occurred with 100 μl isopropanol + HCl (110 ml Isopropanol + 330 μl HCl) for 30 minutes. The mentioned setting was used for the cells F98, U87 and TN22. Primary neurons and astrocytes were seeded into 12-well plates and treated at day 44. After 72h cells were incubated with MTT solution as described earlier. After 4h cell lysis was performed with 200 μl isopropanol + HCL. 90 μl of this solution was transferred into 96-well plate for measuring with the microplate reader. The optical density of each well was measured using the microplate reader Tecan Infinite F50 (Crailsheim, Germany) set to 550 nm (wavelength correction set to 690 nm) using i-control software. Cells without drug treatment were used as control. The viability of the different drug treatments is expressed as the percentage of control.

The cell viability assay was performed using 3(4,5 dimethylthiazol) - 2,5 diphenyltetra-zolium (MTT) assay according to [33 (link)] the cell viability was measured. 3000 cells/well were plated in 96 well - plates one hours prior to the drug treatment. On the fourth day cells were incubated with MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) for 4 h at 37°C, 5% CO₂. The lysis of the cells occurred with 100 μl isopropanol + 0.1 N HCl. The optical density of each well was determined using the microplate reader Tecan Infinite F50 (Crailsheim, Germany) set to 550 nm (wavelength correction set to 690 nm) using Magellan software. Plates were normally read within 1h of adding after lysis. Control was cells without drugs. The viability of the cells was expressed as the percentage of control. Assays were performed on at least three independent experiments.