## 1 Introduction
### IBCA for Monitoring of Caco-2 Cells Membrane Integrity and Viability in Real-Time  
Due to the aforementioned limitations of the TEER method, we decided to apply IBCA performed in the xCELLigence real-time cell analyzer (RTCA) S16 (Pilot Scale) purchased from Agilent Technologies, Inc. (Santa Clara, CA, USA) in order to optimize a dynamic monitoring of the Caco-2 cell kinetics, which form the population of fully differentiated cells ready for non-invasive real-time investigation of their morphology, membrane integrity as well as viability. The xCELLigence RTCA S16 is the smallest member of the xCELLigence family. The instrument consists of a single 16-well E-plate analyzer (placed inside the incubator at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\)), a computer with RTCA integrated software pro 2.8.1 (Agilent Technologies, Inc., Santa Clara, CA, USA) and the single-use sterile E-plate 16 PET (polyethylene terephthalate) (Agilent Technologies, Inc., Santa Clara, CA, USA), which consist of 16 wells with gold plated electrodes integrated into the bottom of wells. According to the website ([[http://www.agilent.com](http://www.agilent.com)]([http://www.agilent.com](http://www.agilent.com)), accessed: 25 February 2025), the other devices from xCELLigence family offered by Agilent Technologies include the following: xCELLigence RTCA SP (Single Plate) or MP (Multiple Plates) used for cell health characterization, immune cell-mediated killing, viral cytopathic effects, cytotoxicity, cell adhesion, cell barrier function and cell signaling; xCELLigence RTCA eSight--Imaging & Impedance, which combines label-free impedance technology with bright-field and fluorescence imaging; xCELLigence RTCA DP (Dual Purpose) instrument, which continuously measures cell invasion and migration (CIM) and monitors cell health as well as behavior; xCELLigence RTCA HT (High Throughput Model) integrated with BioTek BioSpa 8 automated incubator instrument for high throughput screening of therapeutic antibodies, drug compounds, and cytotoxicity on 384-well electronic microplate (E-Plate 384); and last but not least the most advanced xCELLigence RTCA CardioECR (Cardiomyocyte Contractility & Electrical Activity) dedicated for assessment of cardiomyocyte contractility, viability, and electrophysiology. The gold microelectrodes in E-Plate 16 PET inserted into xCELLigence RTCA S16 allow for a dynamic monitoring of cell adhesion, spreading, proliferation, attachment quality with the substrate, morphology, and cell-cell interaction properties. They are submersed in an electrically conductive solution, and the current flows under the working microelectrode. An electric circuit is said to be complete when an electric potential is applied across the biosensors; the electrons exit from the negative terminal, pass through the culture medium, and deposit onto the positive terminal. The weak current applied can flow through the cell-cell junctions, between cell and substrate, and through cell membranes. As the cells adhere to the surface and begin to grow over time, a relative change in electric impedance is observed due to the electrochemical properties of the cells (Figure 1A). Upon reaching confluence, the cells form a plateau, and changes in impedance are recorded and converted automatically into a unitless and dimensionless parameter called cell index (CI) [14]. A zero CI means there is no attachment of cells; however, a higher CI reflects increased cell adhesion to the plate. The current and voltage applied through the electrodes are ~1 uA and >10 mV, respectively. Neither the gold microelectrode surfaces nor the applied electrical potential have effects on cells health, behavior, and metabolism. When a small change occurs in cells' growth, the xCELLigence RTCA S16 automatically and accurately measures the corresponding changes in impedance and converts the analog electronic signals to digital signals that represent information required for calculating multiple biological parameters and for processing the data and plotting graphs.