Incucyte 2021a

Five thousand, 7500 or 10,000 cells per 96-well plate, 55,000 cells per 24-well plate or 300,000 cells per 6-well plate were seeded 24 h in advance, respectively. For KEAP1 siRNA knockdown, 20,000 cells were seeded in a 24-well plate on top of the transfection mix and incubated for 48 h followed by treatments for 24 h. Upon treatment, (Ferrostatin-1 [1 or 5 µM], RSL3 [0.1 µM or 1 µM], iFSP1 [10 µM], Erastin [0.37 µM], Sulfasalazine (SAS) [0.17 mM], Imidazole Ketone Erastin (IKE) [1.11 µM], ML210 [0.37 µM], ML162 [1.11 µM], TBHQ [25 nM]) cells were imaged using the 10× objective within the IncuCyte live cell imager (Sartorius). For dead cell quantification, 100 nM DRAQ7 (Thermofisher) were added to each well. For lipid ROS determination, cells were stained with 5 µM BODIPY C11. Cells were imaged for indicated timepoints every 2 h. Analysis for confluence, DRAQ7-positive (dead) or BODIPY C11-positive cells was performed using the Software IncuCyte 2021A (Sartorius).

To form spheroids, approximately 1000 cells per well were seeded of different carcinoma cell lines into U-bottom 96-well plates (Greiner Bio-One, Frickenhausen, Germany) in their respective media. Brightfield and fluorescence images were taken every 12 h using a Satorius Incucyte SX5 live cell imaging system. Numeric evaluation was performed with the Incucyte spheroid software (Incucyte 2021A, Sartorius, Germany).