Oregon green 488 bapta 1 am ogb 1

For imaging studies of functional Ca²⁺ activity in primary hippocampal cultures, we used a Zeiss 510 NLO fluorescent confocal microscope (Carl Zeiss, Germany) with a W Plan-Apochromat 20 × /1.0 objective. This method allows visualization of the functional neural network architecture at the cellular level. Oregon Green 488 BAPTA-1 AM (OGB-1) (0.4 μM, Thermo Fisher, United States), which was used as a calcium sensor, was dissolved in DMSO (Sigma, Germany) with 4% Pluronic F-127 (Thermo Fisher, United States) and then added to the culture medium for 40 min at 37°C and 5% CO₂. OGB-1 was excited at 488 nm and recorded in the range of 500–530 nm. Time series of 512 × 512 pixel images of 420 × 420-μm fields of view were recorded at 2 Hz. A confocal pinhole of 1 airy unit was used to obtain an axial optical slice resolution of 1.6 μm. Detection and further analysis of Ca²⁺ oscillations was performed in the Astroscanner program. A more detailed description of the image analysis is provided in our previous articles (Vedunova et al., 2013 (link); Zakharov et al., 2013 (link)). The following parameters of spontaneous Ca²⁺ activity were taken into account: the percentage of functional active cells and the duration (s) and frequency (the amount of Ca²⁺ events/min) of Ca²⁺ oscillations.

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 (link)].

The functional metabolic activity of cells in primary neuronal cultures was studied according to the calcium imaging technique. Spontaneous calcium activity of cells was recorded using a calcium-sensitive dye Oregon Green 488 BAPTA-1 AM (OGB-1) (0.4 mM, Thermo Fisher, Waltham, MA, USA) and Zeiss 800 LSM confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). OGB-1 was excited at a wavelength of 488 nm; fluorescence emission was recorded in the rage of 500–530 nm. The resolution of the obtained image was 512 × 512 pixels, the size of the field of view was 420 × 420 µm, and the image recording frequency was 2 Hz. Detection and analysis of calcium oscillations were performed using the Astroscanner program [56 (link),58 ]. The following parameters were assessed: percentage of cells exhibiting Ca²⁺ activity (%), the duration (time from the beginning to the end of an oscillation, s) and frequency (average number of oscillations per min) of Ca²⁺ oscillations.

For calcium imaging, the preBötC was stained by Oregon Green 488 BAPTA-1 AM (OGB-1, Thermo Fisher Scientific Inc., Waltham, MA, United States) as described in detail previously (Winter et al., 2009 (link)). Briefly, 50 μg of OGB-1 was dissolved in 40 μl of DMSO containing 20% Pluronic F-127 (Thermo Fisher Scientific Inc., Waltham, MA, United States), and stored at -20°C in 4 μl aliquots before use. One aliquot of this stock solution was dissolved in 16 μl of an extracellular solution containing the following (in mM): 150 NaCl, 2.5 KCl, 10 HEPES, pH 7.4) to prepare 200 μM of OGB-1 at a final concentration. The OGB-1 solution was injected into the preBötC at the depth of 50–100 μm from the rostral surface of the slice for 10 min under 0.7 bar, followed by a perfusion for >40 min at 28°C to washout excess OGB-1.

Ca²⁺ transients were recorded from single cells across different regions of the plate showing high cellular density at 80–90 days of differentiation, using the cell-permeant Ca²⁺ indicator Oregon Green™ 488 BAPTA-1 (OGB-1 AM; peak absorption = 493 nm; Thermo Fisher Scientific, Carlsbad, CA, United States). Epifluorescence imaging was performed with a mercury arc lamp and using a band pass excitation (450–490 nm) filter. Emitted light was detected with an electron-multiplying CCD camera (High Performance CCD Sensicam, PCO Cooke) with a band pass filter (515–565). Three SZ (#2, 3, and 4) and two HC (#1 and 2) cell lines were analyzed (2-3 plates per cell line; 3–27 neuronal aggregates per plate). The loading solution consisted of OGB1 3.2 µM, Cremophor EL (Merck, Darmstadt, Germany) 0.01% v/v and Pluronic F-127 (Merck, Darmstadt, Germany) 0.4% in NDM. Cells in the loading solution were incubated for 1 h in the dark at 37°C and 7% CO₂. After washing twice with NDM, the medium was replaced with ACSF, and cells were maintained for 30 min before imaging. The regions of interest were recorded for ∼4.7 min at 6.64 Hz (T = 0.1506 s; 1877 frames in total). TTX (0.2 µM) was added to the bath to confirm the AP-dependence of Ca²⁺ transients.