## 3 Results
### Cell Sorting and Imaging Validate the Effects of GSH on Cell Phenotype and Adherence Properties  
In order to identify the cellular effects involved, 13s24 cells were exposed during 6 h to several doses of GSH. Adherent cells retrieved by dissociation were analyzed by cell sorting (Figure 4).  
The adherent cell number was not significantly affected in cell cultures treated with 2.5 mM, and only slightly with 5 mM, suggesting that glutathione does not affect cell survival at physiological concentrations. In flow cytometry, the forward-scatter light intensity (FSC) is proportional to the diameter of cells. After 6 h of treatment with GSH, cell size was significantly increased in a dose-dependent manner (Figure 4C). Cell adhesion force is under the control of actin polymerization (F-actin), allowing for intracellular cytoskeleton stability [22]. A TRITC-tagged phalloidin, which specifically interacts with F-actin subunits, was used to detect modifications of adhesion strength. Figure 4D shows a significant reduction in fluorescent phalloidin incorporation in cells treated during 6 h with GSH 2.5 and 5 mM, indicating a decrease in F-actin. Since the effect of actin polymerization reflects significant modifications to the cell cytoskeleton and is related to extracellular matrix organization, we then investigated whether GSH may affect extracellular proteins involved in cell adhesion. Most of them are highly glycosylated; therefore, their extracellular addressing was quantified using extracellular binding to a lectin, concanavalin A (ConA), tagged with Atto488 (detected by the FITC filter), then compared to the whole cell content of glycosylated sites using an Alexa Fluor 584-coupled ConA detected by the TRITC filter (Figure 5). Extracellular distribution of ConA quantified by cell sorting was down  
Figure 4: Cell sorting analysis of 13s24 adherent cells 6 h after treatment with reduced glutathione (GSH) by flow cytometry. (**A**) Adherent cell counts; (**B**) control cells labelled with phalloidin–TRITC indicate the fluorescent population (gate) with central bracket indicating the mode of distribution (**C**) Cell size (forward-scatter—FSC); (**D**) phalloidin–TRITC fluorescence intensity in response to increasing GSH concentrations (n = 6). (**E**) Representative scatter plots with quartiles (Q). Different letters on the graphs indicate statistical differences at \(p<0.05\) (ANOVA, Fisher’s PLSD).  
regulated in a dose-dependent manner after exposition to GSH over 6 h (Figure 5D). This result suggests that GSH modifies cell size and/or adhesion by modulation of extracellular proteins associated with a loss of F-actin polymers.  
Figure 5: Reduced glutathione (GSH) affects glycoprotein on the cell surface in a dose-dependent manner. (**A–D**) Cell sorting analysis of 13s24 cells 6 h after treatment with several doses of GSH detected with extracellular binding with either concanavalin A (ConA)-Atto488 or ConA-AlexaFluor594 (ConA Total) (**A**). (**B**) Representative histogram of ConA Total cell distribution according to labelling intensity and the selection of fluorescent population (gate) with central bracket indicating the mode of distribution (**C**) Representative distribution of ConA Extracellular versus ConA Total in quartiles (**Q**) according to GSH concentrations. (**D**) Data are presented as regression curve using mean values of ConA-Atto488 fluorescence intensity \(\pm\) SEM (6 replicates). (**E**) Cell imaging at objective \(\times\)20 of 13s24 cells 6 h after treatment with GSH 10 mM, labeled extracellularly with ConA-Atto488 (green) then, after permeabilization, with ConA-AlexaFluor 594 (red) and Dapi (blue).