Fluo-8 AM for Intracellular Ca2+ Oscillations

A calcium indicator Fluo-8 AM was used to detect the intracellular Ca²⁺ oscillations as previously described 23 (link) with modifications. Cell samples including the Control, Control+MSC-Exo, LPS, and LPS+MSC-Exo groups (n=5 each group) were washed in serum- and phenol red-free DMEM containing 4 μM Fluo-8 AM (Abcam, ab142773, California, USA) plus 0.08% Pluronic F127 (Life Technologies, California, USA) for 20 min at 37°C, 5% CO₂ to load the dye into the cells. Next, cultures were washed thrice and stored in artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 25 mM NaHCO₃, 2.5 mM KCl, 1 mM KH₂PO₄, 2 mM CaCl₂, 2 mM MgSO₄, and 10 mM glucose. Resting Ca²⁺ levels were recorded in ACSF for 30 s, and then 10 mM adenosine monophosphate (ATP) was used to stimulate the Ca²⁺ influx. The fluorescence of Fluo-8 AM was excited at the wavelength of 488 nm and measured every 1 second for 180 s using a confocal microscope (Olympus, FV3000, Japan). Calcium influx and resting Ca²⁺ levels were measured in individual astrocytes using the image analysis software Cellcens (Olympus, Japan). More than 90 cells for each experimental condition were analyzed using Igor Pro software (WaveMetrics, Oregon, USA) and results from at least three independent experiments were averaged. The intensity of excitation light and sampling frequency were kept as low as possible to minimize bleaching.