## 4 Discussion
\begin{table}
\begin{tabular}{p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt}} \hline \hline
**Cell Growth Phase** & **Cell Adhesion** & **Differentiation** & **Confluency/ Plateau** & **Physiological Cell Death** & **Fluctuations upon Addition of Fresh Media** & **Fluctuations upon Addition of Addition of L-DOPA** \\ \hline
**Observed CI or changes in CI** & Between 0–1.0 & Start: 0.5–1.0 End: 4.0–8.5 End: 4.0–8.5 End: 8.5 & Start: 4.0–8.5 End: 0.0–0.5 & max. \(\pm\) 2.0 (\(\pm\) 40\%) & max +2.0 (+40\%) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Changes in observed CI values depending on the cell-growth phase. Furthermore, membrane integrity (i.e., the presence of a functional, polarized monolayer) was tested with the use of TNF-\(\alpha\) (100 ng/mL), and the results were compared to the classic TEER method [20]. However, no significant decline in electrical impedance--expected as a result of the TNF-\(\alpha\) effect on TJs and membrane integrity of Caco-2 cells--was observed using the xCELLigence RTCA S16 (Figure 6). Thus, the used device is inadequate for the assessment of the integrity of a polarized Caco-2 cells' monolayer as compared to the TEER method. Another disadvantage of the xCELLigence RTCA S16 is that the system does not allow for compartmentalization into distinct apical and basolateral regions of the monolayer for polarization studies. Our final experiment involved incubation with 1% FBS and treatment of fully differentiated Caco-2 cells with a high dose of L-DOPA (100 ug/mL). According to the literature, this concentration has been reported to be toxic to undifferentiated Caco-2 cells [22]. This effect was similar in our in-house MTS cytotoxicity test (Figure 7). Our next real-time experiments also confirmed the cytotoxic effect of L-DOPA on Caco-2 cells cultured for at least 17 days post-seeding. Based on these findings, we propose that the described protocol using the xCELLigence RTCA S16 system is suitable for real-time assessment of the effects of test compounds on the viability of fully differentiated Caco-2 cells. Yet, the mechanisms of action on enterocytes of the abovementioned compounds, namely TNF-\(\alpha\) and L-DOPA differ, too. TNF-\(\alpha\) is known for its strong proinflammatory potential and to disrupt tight junctions [24]. TNF-\(\alpha\) impairs the TJ function by activating the NF-\(\kappa\)B pathway and reducing ZO-1 production [25]. For differentiated Caco-2 monolayers, using TNF-\(\alpha\) around 10-100 ng/mL is typical for permeability or junctional disruption studies, and only partial toxicity can be expected at \(\sim\)300 ng/mL [26]. L-DOPA, due to its auto-oxidation to highly reactive quinones and semiquinones as well as reactive oxygen species, can cause damage to cellular components (including mitochondria) and lead to cell death in vitro, especially in non-dopaminergic neurons [27]. Furthermore, due to the fact that Caco-2 cells can convert L-DOPA to dopamine (DOPA decarboxylase is present and measurable) [28], auto-oxidation can also lead to the formation of dopamine-derived quinones and subsequent mitochondrial membrane depolarization, ATP synthesis inhibition, and caspase-mediated apoptosis [29]. Differentiated Caco-2 cells exhibit key characteristics like the formation of tight junctions, microvilli, and expression of enterocyte-specific proteins, all of which contribute to a functional intestinal barrier. Undifferentiated Caco-2 cells express very low levels of enterocyte-specific enzymes [30], are characterized by decreased expression of intestinal transporters [31], and thus are more prone to toxicity, which could explain the difference in the cellular index in differentiated vs. undifferentiated Caco-2 cells treated by a high dose of L-DOPA on E plate 16 (Figures 8 and 9). In summary, the TEER method is a valuable tool for studying the integrity and function of cellular barriers, while the xCELLigence RTCA S16 system can provide a continuous assessment of cell behavior in response to various stimuli (Table 2).