## 2 Results  
### SeNrs Induce the Generation of Ca\({}^{2+}\) Signals in Cortical Astrocytes  
Previously, we found that nanoselenium in the form of spherical particles dose-dependently causes the generation of Ca\({}^{2+}\) signals in astrocytes of various parts of the brain [27], and the EC50 for nanoparticles depended on their size [28]. In this study, application of SeNrs to cortical astrocytes led to the generation of Ca\({}^{2+}\) responses starting from a concentration of 0.5 \(\upmu\)g/mL (Figure 1A). Ca\({}^{2+}\) signals of astrocytes are characterized by the generation of transient Ca\({}^{2+}\) responses, followed by a gradual increase in [Ca\({}^{2+}\)]i and the establishment of a new steady state [Ca\({}^{2+}\)]i at concentrations of 5-10 \(\upmu\)g/mL. Lower concentrations of SeNrs caused a gradual increase in [Ca\({}^{2+}\)]i after a lag period of varying duration. An analysis of the dependence of the amplitude of the Ca\({}^{2+}\) signal on the addition of SeNrs on the concentration of nanorods showed an increase in the level of [Ca\({}^{2+}\)]i with an increase in the concentration of SeNrs and EC50 by 4.9 \(\pm\) 0.4 \(\upmu\)g/mL (Figure 1B). To generate Ca\({}^{2+}\) signals, cells can use both the entry of Ca\({}^{2+}\) ions from outside and the mobilization of Ca\({}^{2+}\) from intracellular pools. The addition of 5 \(\upmu\)g/mL SeNrs in a nominally calcium-free medium in the presence of 0.5 mM EGTA caused the generation of Ca\({}^{2+}\) signals in astrocytes (Figure 1C), with an amplitude comparable to the addition of SeNrs in the standard medium (Figure 1A). Emptying the Ca\({}^{2+}\) pool of the endoplasmic reticulum (ER) by applying 10 \(\upmu\)M thapsigargin (TG) prevented the generation of astrocyte Ca\({}^{2+}\) signals in response to the addition of SeNrs (Figure 1D). Thus, SeNrs dose-dependently induced the generation of Ca\({}^{2+}\) signals in the form of a gradual increase in [Ca\({}^{2+}\)]i in cerebral cortex astrocytes due to the mobilization of Ca\({}^{2+}\) ions from the thapsigargin-sensitive pool of the endoplasmic reticulum. Figure 1: _Cont._  
SeNrs Protect Cortical Astrocytes from OGD-Induced Death by Suppressing the Global Increase in Cytosolic Calcium Concentration  
Preincubation of astrocytes with 1 or 5 \(\upmu\)g/mL SeNrs for 24-h did not cause cell death (Supplementary Figure S1). Ischemia-like conditions (OGD) lasting 40 min induced a biphasic increase in [Ca\({}^{2+}\)]i in astrocytes (Figure 2A, red curve). This correlated with the appearance of propidium iodide (PI) fluorescence after OGD (Figure 2B--OGD), which is a sign of necrotic cell death (Figure 2C). After a 24-h preincubation of astrocytes with 1 \(\upmu\)g/mL SeNrs, there was a significant inhibition of the OGD-induced phase of the global increase in [Ca\({}^{2+}\)]i (Figure 2A, green curve). In addition, the number of PI-stained cells decreased (Figure 2B--SeNrs (1 \(\upmu\)g/mL + OGD), which may indicate suppression of necrosis (Figure 2C). Increasing the concentration of SeNrs to 5 \(\upmu\)g/mL led to complete inhibition of the OGD-induced phase of the global increase in [Ca\({}^{2+}\)]i (Figure 2A--orange curve) and suppression of necrosis (Figure 2B,C). Thus, preincubation of astrocytes with SeNrs did not inhibit the first reversible phase of the OGD-induced [Ca\({}^{2+}\)]i increase, but suppressed the phase of the global [Ca\({}^{2+}\)]i increase, when 5 \(\upmu\)g/mL SeNrs completely suppressed these astrocyte Ca\({}^{2+}\) signals. Inhibition of the global increase in [Ca\({}^{2+}\)]i led to a decrease in the number of astrocytes dying due to necrosis. Figure 1: SeNrs dose-dependently cause the generation of Ca\({}^{2+}\) signals in cortical astrocytes through the mobilization of Ca\({}^{2+}\) ions from the thapsigargin-sensitive pool of the endoplasmic reticulum. (**A**) Ca\({}^{2+}\) signals of astrocytes to the addition of various concentrations of SeNrs. Ca\({}^{2+}\) responses of astrocytes to SeNrs averaged over several tens of cells are presented.