Ab120873

Astroglia were plated in 96-well plates with black walls and a clear base (Costar, Glendale, Arizona, USA) at a concentration of 20,000 cells per well, and were cultured in medium containing 100 µM INI-0602 for 40–60 min at 37 °C. Untreated or INI-0602-treated astroglia were then incubated with 2 mM Fura-2 AM (ab120873; Abcam) in HBSS for 40 to 60 min at 37 °C. Following this manipulation, buffer was extracted using a multi-pipette, and cells were rinsed for 15 to 20 min using HBSS to wash away extracellular dye. Cells were preheated for 10 min before data collection was initiated. Fura-2 fluorescence was assessed at excitation wavelengths of 340 and 380 nm with an emission wavelength of 520 nm, using a Flexstation 3 plate reader (Molecular Devices, San Jose, CA, USA). A sampling interval of 3 s with 60 reads per well was applied. The Fura-2 ratio was computed using Softmax Pro v5.4 (Molecular Devices), and reactions were quantified by calculating the AUC.

The Rhod-2 AM dye (4 µM, 20 min, ab142780, Abcam, UK) and Fura-2 AM dye (2 µM, 20 min, ab120873, Abcam, UK) were used to detect the mitochondrial and cytosolic Ca²⁺ level, respectively. Images were obtained every 10 s, and Ionomycin (50402ES03, Yeasen, China) was added 60 s after the beginning of the observation [39 (link)]. The relative fluorescence intensity was measured by Image J software. OCR of hepatocytes was measured by using the XF24 Extracellular Flux Analyser (Agilent SeaHorse Bioscience, USA) as described previously [40 (link)].