Figure 5

Effect of carnosine on spheroid size and cell viability. (A) Images of spheroids cultured in the presence and absence of 20 mM carnosine. Carnosine was added after spheroids had already formed (upper image) or before spheroid formation (lower image). The images show the same time points of spheroid growth, although the duration of carnosine treatment differs as indicated, depending whether carnosine was added before or after spheroid formation. pH values represent measured pH of media collected from all respective spheroid samples (N > 30) at the end of the experiment. (B) In both experimental conditions, the diameters of carnosine-treated spheroids were significantly smaller than those of controls (t-test, **p < 0.01). (C) Flow cytometry showed that treatment with different concentrations of carnosine (5–40 mM) decreased cell viability in hypoxic conditions in a dose-dependent manner (t-test, *p < 0.05, **p < 0.01). Numbers in the columns give the ratio for the viability of a carnosine-treated sample and the respective hypoxic control (set as 100%). The ratio of the viability of hypoxic and normoxic controls was 96%. (D) Cultivation of HeLa spheroids in the presence of 20 mM carnosine significantly reduced cell viability by approximately 50% compared with the control group (t-test, **p < 0.01).