## 4 Discussion
It is not always easy to obtain the same cell density when the cell supports are different: although there is not significant difference between E-96 and 96 well plates, well size and bottom glass of Ibidi dishes have significant impact on cell proliferation. Besides the comparison of the different methods, it is of interest to comment the effects of GSH on 13s24 cells. The most significant result of this work is the dose-dependent alteration of adhesion strength and increase of olfactory neuron cell size. The dose-response of cell index (xCELLigence) was strongly correlated from control conditions to 10 mM GSH (r\({}^{2}\) = 0.9963, Figure 1) without significantly affecting cell proliferation and survival (Figure 4A). Such high GSH concentration has been reported in liver cells [3]. The decrease in cell index without affecting cell number suggests an effect on cell adhesion strength. GSH-induced decrease in cell motility (4D), reduction in F-actin content (Figure 4C), and extracellular glycosylation sites (Figure 5) suggest that GSH reduces cell force adhesion to the extracellular matrix (ECM). Indeed, the regulation of cell adhesion, required for the control of cell growth, migration, as well as differentiation, involves a fine regulation of the cytoskeleton by controlling actin polymerization (F-actin) by integrins, which are highly glycosylated proteins [27]. Moreover, GSH was found to increase neuroblast cell size in a dose-dependent manner (Figures 3B and 4B). GSH is produced in the cytosol of most cells [3], and several studies have shown that they are able to export GSH. Two types of carriers were identified: protein multidrug resistance protein (MRP) [28] and organic anion transporting protein (Oatp1) [29]. Cells produce GSH in their cytosol; that GSH can be exported to the extracellular space and can react with extracellular components. Cell adhesion is regulated by complex assemblages of integrins, which are highly glycosylated proteins containing disulfide bonds in the extracellular domains. The binding capacity of integrins can be modulated by various stimuli; the disulfide bonds maintain the integrin in a less active form. The addition of reducing agents results in increased ligand binding [30]. Conformational regulation of integrin affinity (\(\alpha\)4\(\beta\)1) by reducing agents has been demonstrated in many studies [31]. GSH regulates integrin \(\alpha\)2\(\beta\)3-mediated cell adhesion [32]. Similarly, the disruption of disulfide restriction at integrin knees induces activation and ligand-independent signaling of \(\alpha\)4\(\beta\)7 [33]. All these observations strengthen our hypothesis that GSH produced in cell cytosol can be exported to interact with integrins, giving the cells an opportunity to control their volume. One may argue that the GSH concentrations tested in the present study were not physiological. Admittedly, the plasma GSH concentration is low (in the order of \(\mu\)M), but intracellular concentrations are in the millimolar range and can even reach 10 mM in liver cells; much of it is exported across the plasma membrane into extracellular spaces [34]. It must be noticed that GSH is a highly reactive compound. It is then not a surprise to detect low concentrations of free GSH in the interstitial liquid. Ultimately, what matters is the turnover rate of GSH. GSH turnover rates in most cells are rather fast, with half-lives between 2 and 6 h [35; 36], indicating high rates of both GSH synthesis and export [3]. In conclusion, despite rather low concentrations of GSH, high turnover rate and high cell sensitivity are compatible with the role of GSH in the regulation of cell volume. Further, our results already show an increased cell volume at 1 mM GSH, which, according to the literature, seems common in cells [1; 14]. In a previous study, we observed a similar effect of GSH on human lung epithelial cell volume suggesting that the effect of GSH on cell volume is a common feature. We also showed that the effect of extracellular GSH is counterbalanced by hydrogen peroxide [14].