Determination of the IC50 by applying the MTT cell viability assay

The MTT assay was performed as described in David Morgan's “Polyamine Protocols. Methods in Molecular Biology”³³ with minor changes of the protocol as described: 24 h after plating, 50% confluent cells were treated in concentration series including the expected IC₅₀ concentration of the respective substance. After 72 h of incubation the MTT reaction and photometric analysis was implemented. The cell culture medium was aspirated with a vacuum pipette before applying 100 µL MTT working solution (MTT stock solution: 20 mg/ml MTT (Sigma, Cat. No. M5655) in 1× PBS (D8537, Sigma); MTT working solution: 1:5-dilution of the stock solution with RPMI 1640 medium without phenol red). The reaction was stopped after 2 h at 37 °C with 100 µL isopropanol (70 %, Cat. No. 3889017). The photometric analysis at λ = 560 nm was performed in a TECAN Plate Reader after an additional 30 min incubation at room temperature (RT). The viabilities were normalized to DMSO controls. For the identification of the IC₅₀ values all experiments were performed in three replicates and repeated in at least three independent experiments.