*2.2. Reactive Oxygen Species Concentration*

The DCF-DA test was used to predict extracellular ROS accumulation after 1 h or 24 h incubation with glucose (50–150 mM) and insulin (50–250 μM) (Figure 3).

**Figure 3.** ROS levels in neuron-like cells after: (**A**) 1 h incubation with glucose or insulin and (**B**) 24 h incubation with glucose (50, 100 or 150 mM) or insulin (50, 100 or 150 μM). Control—untreated neuron-like cells; H2O2—cells incubated with 50 μM H2O2 (positive control). Statistically significant differences compared to the untreated neuron-like cells: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

After 1 h incubation, a statistically significant increase in the level of ROS was observed both in the presence of glucose and insulin. A statistically significant increase in ROS levels was observed after 1 h incubation with glucose or insulin in all analyzed concentrations, with the highest concentration obtained for 150 mM glucose (x-fold—1.2). After insulin administration, the highest level of ROS was obtained after 100 μM (x-fold—0.8). No concentration dependence was observed 1 h or 24 h after glucose or insulin administration.

It is worth noting that the impact of glucose and insulin on ROS levels was less expressed after 24 h incubation, as there was an ample decrease in the level of free oxygen radicals. Furthermore, these data were supported by the results obtained with positive assay control (H2O2), confirming the observation that reactive oxygen species are the first messengers of stress in the cells, and their levels are diminished after 24 h of treatment.
