L5 filter set

The primary neuroglial culture was obtained as described above and divided into two groups 4 h after the cells were attached. First group was treated by MSC-EV at a concentration of 2.4 × 10⁸ particles/mL, while the control group was treated with vehicle and the culture medium was not changed during the entire experiment. The analysis of cell cultures was carried out daily for 2 plates from each group during 7 days, after which they were not used. Hippocampal cell cultures were loaded with a Calcein-AM probe at a final concentration of 5 µM for 40 min at 37 °C in HBSS with 10 mM HEPES followed by a triple wash with HBSS. Cell slips were mounted in an experimental chamber and observed using a Leica DMI6000B fluorescent inverted motorized microscope with a HAMAMATSU C9100 high-speed monochrome CCD camera. For excitation and recording of Calcein fluorescence, a L5 filter set (Leica, Germany) was used, containing excitation filter BP480/40, beam splitter FT-505 and emission filter BP527/30, with a Leica EL6000 excitation light source containing a HBO 103 W/2 high-pressure mercury lamp. Neurite length analysis was performed using the Image J software.

For recordings of changes in mitochondrial or cytosolic ROS production, hippocampal cell cultures were loaded with MitoSOX Red (mitochondrial ROS indicator, 5 µM, 15 min incubation; 37 °C) or H₂DCF-DA (mainly cytosolic ROS indicator; 10 µM, 20 min incubation; 37 °C). After incubation with the dyes, cells were washed three times before the experiment. To measure the ROS generation, we used the system based on the inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100 and a high-speed light filter replacing system Leica’s Ultra-Fast Filter Wheels with replacing time 10–30 ms. For excitation of DCFH₂-DA and MytoSOX Red we used L5 filter set (Leica, Germany) with excitation filter BP480/40, dichroic mirror 505 and emission filter 527/30. We determined the shape ROS production rates under oxygen-glucose deprivation (OGD).

For ROS production detection, cortical cell cultures were stained with H₂DCF-DA (10 µM, 20 min incubation; 37 °C) and washed three times before the experiments. To measure the ROS production, we used the inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100 and a high-speed light filter replacing system Leica’s Ultra-Fast Filter. For excitation of DCFH₂-DA, we used the L5 filter set (Leica, Germany) with excitation filter BP480/40, dichroic mirror 505, and emission filter 527/30. We determined the shape of ROS production rates under oxygen-glucose deprivation (OGD).