## 4 Discussion  
The purpose of this work was to optimize preparation of a Caco-2 monolayer with fully differentiated cells ready for real-time investigation of epithelial barrier and viability by the xCELLigence RTCA S16 device. Although the xCELLigence real-time system was already used to study the integrity of the Caco-2 membrane, the data provided by the authors of this study were insufficient--i.e., no information about total post-seeding incubation time of Caco-2 cells, number of seeded cells, exact time of media change, or type of used E-plate were given [16]. Here we propose a complete and replicable protocol to study fully differentiated Caco-2 cells cultivated on E-plate 16. Cells should be seeded in the concentration of 2000 cells per well in cell culture medium supplemented with 20% fetal bovine serum (FBS) until the proliferation state is reached. Next, the old media should be replaced by fresh cell culture medium supplemented with 1% FBS. Cells should be incubated at least 17-18 days post-seeding to be differentiated and ready for experiments, as it is recommended in the literature [12]. The real-time analysis measured by xCELLigence system showed that long and stable horizontal line of the confluent cells was reached (Figure 5A). What is more, the staining procedure successfully confirmed the expression of occludin, a transmembrane protein that regulates the permeability of epithelial barriers [21], and, thus, the presence of TJs in Caco-2 cells cultivated on E-plate 16 (Figure 5D). The cells also showed a high cellular index (CI) and were ready for subsequent/other experiments after around 17-18 days post-seeding. According to our observations, a decrease in the CI curve was directly related to their death, detachment, and change in shape, which could be clearly observed under a microscope. Thus, we confirmed that the xCELLigence Real-Time Cell Analyzer (RTCA), a non-invasive, impedance-based biosensor system can indeed measure Caco-2 cells' viability, growth, proliferation, and differentiation. The CI changes in time were summarized in Table 1.  
The major advantages of this system include the following: rapid, effortless, and continuous detection of cell kinetics and quality attachment in real-time with high sensitivity, accuracy, and reproducibility as compared to the standard TEER protocol. One should remember that since RTCA uses microelectrodes embedded in a cell culture plate to measure changes in electrical impedance as cells attach, grow, and interact with the surface, measurements can be affected by electroactive compounds added to the media. Thus, according to our observations, the tested substances should be added carefully, directly to the old media on E-plate 16, in order to avoid the fluctuation of CI. It should also be mentioned that according to the manufacturer's recommendation, E-plates are disposable after a single use, yet it was already reported that E-plates can be successfully regenerated and reused several times without significantly affecting experimental results [23]. What is more, we expect that the xCELLigence RTCA S16 system could be used for other epithelial cell models. We recommend starting the optimization procedure as described above, namely: (1) selecting the appropriate cell seeding density; (2) monitoring the CI (cell index) curves, with particular attention to the duration of the plateau phase--where most experiments are typically conducted; and (3) reducing the FBS concentration (if necessary) during the mid-proliferation stage.