*4.4. Calcium Imaging*

The functional calcium activity of astrocytes was studied using an LSM 510 laser scanning microscope (Carl Zeiss, Oberkochen, Germany) with a W Plan-Apochromat 20×/1.0 objective. The calcium imaging technique allowed visualization of the functional architecture of cells in culture. We used the fluorescent calcium-sensitive dye Oregon Green 488 BAPTA-1 AM (OGB-1) (0.4 μM, Thermo Fisher Scientific, Waltham, MA, USA) dissolved in dimethylsulfoxide (DMSO) (Merck KGaA, Darmstadt, Germany) with 4% Pluronic F-127 (Thermo Fisher Scientific, Waltham, MA, USA). OGB-1 was added to the culture medium and incubated for 40 min in a CO2 incubator. The fluorescence of OGB1 was excited at 488 nm by argon laser radiation, and emission was recorded in the range of 500 to 530 nm. The dynamics of intracellular calcium concentration were measured by analysis of a time series of 512 × 512 pixel images capturing 420 μm × 420 μm fields of view that was recorded at 2 Hz. The following parameters of the functional calcium activity were assessed: duration of the calcium oscillations (time from the beginning to the end of an oscillation (s)), frequency of calcium oscillations (average number of oscillations per min), and percentage of working cells (ratio of the number of cells in which at least one oscillation was recorded among the total number of cells (%)) [29,30].
