Co2 module s

Immediately after adding the EVs stained with PKH26 Red Fluorescent Cell Linker or CellTracker™ Deep Red dye to BV-2 cells, live cell imaging was performed using an inverse microscope ZEISS Axio Observer.Z1 (Carl Zeiss AG, Germany) equipped with AxioCam MR Rev3 camera (image size of 1388 × 1040 pixel, bit depth of 12), Plan-Apochromat 63x/1.40 oil objective and live cell incubation setup consisting of Heating Unit XL at temperature of 37 °C and CO₂ Module S at 8 % CO₂ (PeCon GmbH, Germany). Measurements were done in z-stacks with Brightfield and Fluorescence exposure time of each 300 ms, filter set no. 50 (Carl Zeiss AG) for Cy5, excitation filter at 625–655 nm, emission filter at 665–715 nm and dichroic mirror at 660 nm for EVs with CellTracker™ Deep Red dye as well as brightfield exposure time of 80 ms, fluorescence exposure time of 220 ms, filter set no. 43 (Carl Zeiss AG) for DsRed, excitation filter at 538–562 nm, emission filter at 570–640 nm and dichroic mirror at 570 nm for EVs with PKH26 Red Fluorescent Cell Linker dye.

DIV80 human iPSC-derived astrocytes were incubated with the calcium indicator Fluo-4 AM (1 μM; ThermoFisher Scientific; Cat. No. F14201) for 30 min, followed by two rinsing steps with ScienCell Astrocyte Growth Medium, supplemented with antibiotics, for 10 min each. Time-lapse microscopy was then performed at 0.5 Hz with a Zeiss Axio Observer Z1 Inverted Microscope using 20× magnification (numerical aperture 0.8) and a ZEISS Axiocam 506 mono camera. Temperature (37 °C), humidity, and CO₂ (5%) were maintained at constant levels throughout the course of the experiments through TempModule S and CO₂ Module S devices (PeCon GmbH; Erbach, Germany). LED 488 nm was set at 20% and exposure time was 200 ms to avoid phototoxicity. For spontaneous calcium wave acquisition, recording was conducted for 5 min and focus stabilisation was achieved with the use of Zeiss Definite focus every 30 images. For induced calcium wave acquisition, recording was performed for 1 min, before the addition of 10 μM ATP, followed by an additional 4 min recording. Analysis of at least 25 cells per acquisition video was performed using the calcium signaling analyzer tool, CaSiAn [38 (link)].

Metaphase spreads were visualised with an Imager.Z2 (Zeiss) and imaged with a CoolCube 1 (Metasystems) and analysed using ImageJ. Live-cell imaging was performed on an IX81 (Olympus) fitted with a CO₂ Module S (Pecon) and a JCS controller (Shinko) for CO₂ and temperature control respectively, and imaged with a CoolSnap HQ (Photometrics). Live-cell imaging videos were recorded up to 120 hours. Images were acquired every 5 min. Exposures were as follow: Phase contrast 30 ms, GFP 100 ms and RFP 100 ms. 20× magnification lenses were used for the acquisition of the live-imaging videos.