4.3. Crystal Violet Staining (CytoTox Assay)

The cells were seeded at a minimal amount of 10,000 cells in 100 µL of culture medium per well of a 96-well plate in triplicate and incubated overnight. The cell number per well is mentioned in the legend of the figures showing the experimental results. The following day, the cells were incubated in complete or methionine-free media with or without ligands (TNFα, FasL or TWEAK) or cisplatin (Merck, Darmstadt, Germany) at the indicated concentration. The incubation time is also mentioned in the corresponding figure legend. For staining, the supernatants were removed, and the cells on each well were incubated with 50 µL crystal violet solution (1% crystal violet in 20% methanol; Carl Roth, Karlsruhe, Germany) for 10 min and subsequently washed five times with distilled water. The plates were dried for 24 h in the dark. For quantification, 100 µL of methanol was added to each well, and the plate was incubated for 10 min until the crystal violet was completely dissolved. The photometric absorbance was measured at 595 nm using a microplate reader (Tecan, Crailsheim, Germany). For data analysis, the experiments were repeated three times to calculate the mean values and standard deviations (n = 3). The results were normalized to the untreated control (100%). Therefore, the relative cell number values determined via the crystal violet assay with the stimulated probes (CV_S) were normalized to those of the untreated control (CV_C) ((CV_S/CV_C) = CV_R). To obtain percentage values, the CV_R value was multiplied by 100 (RCN (%) = (CV_S/CV_C) × 100 = CV_R%). For statistical analysis, these results were evaluated with unpaired Student’s t-test. The significance level was set to p < 0.05.