Glass bottom culture dishes

Calcium levels were live monitored in attached astrocytes through the ratiometric probe Fura-2-AM (#F1221, Invitrogen) as done by Kowaltowski et al. (2019) (link). Briefly, cells were plated in glass-bottom culture dishes (#627871, Greiner Bio-One), incubated with 5 μM Fura-2-AM for 30 min at 37°C in experimental medium lacking FBS and supplemented with 1 mg/ml bovine serum albumin (BSA). Fluorescence was assessed at λ_ex = 340 (F₃₄₀) and 380 nm (F₃₈₀) and λ_em = 510 nm in a Leica DMi-8 microscope equipped with a Fura-2 filter (Leica Microsystems). Cells were followed through additions of CGP (10 μM), ATP (100 μM), as well as ionomycin (20 μM) to allow calibration. Analyses were conducted through FIJI ImageJ 1.52p (Schindelin et al., 2012 (link)), in which individual cells (55–125/group per experiment) were identified as regions of interest (ROI) and [Ca²⁺] variation was estimated as the ratio (R) between F₃₄₀/F₃₈₀. Data were calibrated by the maximal ratio induced by ionomycin, controlled for background fluorescence oscillations, and normalized by the initial ratio (R₀).

Embryos or larvae were anesthetized with 0.016% Tricaine-S (MS-222, Pentair Aquatic Ecosystems, Apopka, Florida, NC0342409) in E3 embryo medium. Dissecting microscope fluorescence imaging was performed on a Leica M205FA with a DFC450 C camera and 1.0× PlanApo M-Series objective, illuminated with a TL5000 light base and CoolLED pE-300^white Illumination System. We used the following Leica filter sets for fluorescence: ET GFP 10447408, ET CFP 10447409 and ET DSR 10447412 for imaging and sorting; and TXR LP 10450590 for routine sorting of red fluorescence. Laser scanning confocal microscopy was performed on a Zeiss LSM880 following embedding in E3 with 1% low-melting-point agarose (Sigma Aldrich, A9045) on glass bottom culture dishes (Greiner Bio-One, Kremsmunster, Austria, 627861). Images were collected with a 10/0.8 air-objective lens swith all channels captured sequentially with maximum speed in bidirectional mode, with the range of detection adjusted to avoid overlap between channels. Maximum projections of acquired z-stacks were made using ImageJ/Fiji (Schindelin et al., 2012 (link)) and cropped and rotated using Adobe Photoshop 2022.