## 4 Discussion

Table 1: Comparison of the main characteristics of different real-time measuring devices. (++ and ++ refer to medium and high level skill levels). then required in order to determine which effects are involved, among the regulation of cell number, cell adhesion force, and/or cell surface occupancy (size, differentiation). Another method for monitoring RTCs is the optimization of complementary analyses according to time-dependent events to identify short-term (adhesion, toxicity, etc.) and long-term events (proliferation, differentiation, etc.) involving different signaling pathways [26]. Finally, RTCA data can be monitored and analyzed during experiments, allowing for instant result processing and adjustments to the experiment's duration. The phase holographic cell imaging system HoloMonitor is amazing; the software brings the possibility to follow single cells in time-lapse experiments to quantify their physical (area, perimeter, volume, irregularity, etc.) and behavioral (motility, migration, speed, etc.) characteristics. It is possible to select few tens of cells. It is also possible to select the place to be analyzed inside the wells, which is very convenient in light of cell cultures often being heterogeneous. Such a process takes time before starting acquisition; it may be problematic to study very fast responses, but it is essential to insure efficient measurements. Analysis of the results is time-consuming; therefore, it is necessary to check selected cells from one frame to the following one in order to insure reliable results (Table 1). The third time-lapse method, using the 3D Nanolive microscope, allows for an impressive resolution. It is very user-friendly, and time-lapses bring researchers in the close vicinity of cells. Cell analysis based on refractive index is an original and fascinating approach. Further research is required to increase the potential of the microscope, in particular by defining the refractive index of organelles. The main limitation of the microscopic analysis of cells, apart from the low number of cells analyzed, is that the recent history of cells is unknown, i.e., whether they have divided recently, or whether they will divide soon. Such variations in the metabolic status of cells may introduce confounding results. A limitation applicable to both HoloMonitor and Nanolive is that the time required for processing and sample setup makes it difficult to acquire data quickly enough to measure very early responses (less than 30 min). The different methods show some discrepancies in terms of glutathione's dose-dependent effect. For example, GSH-induced effects were detected from the lower concentration 1 mM in RTCA experiments with xCELLigence (cell index, Figure 1A) and HoloMonitor (Figure 2A), although the effects were detected from 2.5 mM with flow cytometry (Figures 4 and 5). Since flow cytometry measurements require cell dissociation and fixation, decreased sensitivity could result from partial cell and extracellular epitope loss, leading to lower sensitivity than the RTCA and HoloMonitor time-lapse monitoring methods. An important experimental parameter rarely indicated, as seen in Section 2, is the culture media volume in which cells are incubated at the time of measurements. It is usual to refer to the concentrations of molecules tested; however, in the case of molecules having a specific attraction for certain chemical bonds, such as GSH for disulfide bridges, incubating cells in 1 mL represents twice the GSH molecules than in 500 \(\mu\)L. This can lead to significant differences in sensitivities measured by different techniques, especially in 96-well plates (xCELLigence and HoloMonitor) versus Ibidi dishes (Nanolive). In the same order, cell number at the time of measurement may be critically important. Indeed, in the present case, it will change the number of disulfide bridges exposed to GSH.