Fluo 8 am

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.

The calcium indicator Fluo-8 AM (ab142773, Abcam, CA, USA) was used to detect the intracellular Ca²⁺ oscillations in each group of primary culture cells, and the procedures were performed as previously described 18 (link), 24 (link). The cultures were washed in media containing 4 μM Fluo-8 AM for 20 min at 37°C and 5% CO₂ to load the dye into the hippocampal neurons. Next, the samples were washed three times 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). After resting Ca²⁺ levels were recorded in ACSF for 20 seconds, 10 μM adenosine monophosphate (ATP) was used to stimulate Ca²⁺ influx. Fluorescent signals were excited at a wavelength of 488 nm and imaged every 1 second for 120 seconds using a confocal microscope (Olympus, FV3000, Japan). Calcium influx and resting Ca²⁺ levels were measured in individual hippocampal neurons using the image analysis software Cellcens (Olympus, Japan). More than 100 cells for each experimental condition (ΔF/F) were analyzed using Igor Pro software (WaveMetrics, Oregon, USA), and the results from at least three independent experiments were averaged.

A cell-permeable Fluo-8 AM (Abcam, Ltd., Cambridge, UK) was used to evaluate the effect of OH-dDHL on the intracellular calcium mobilization in the BMDMs. The experiments were performed according to manufacturer’s instructions with a minor modification. Briefly, BMDMs were cultured overnight with growth medium in 96-well plates. Then, cells were washed with Hanks’ balanced salt solution to minimize background fluorescence and interference with serum. Cells were incubated with Fluo-8 AM for 1 h in HBSS prior to treatment with OH-dDHL for the indicated dose. The cytoplasmic calcium level was documented by the fluorescence intensity under an SpectroMAX ELISA plate reader (Molecular Devices, San Jose, CA, USA). For the confocal microscopic analysis, cells were seeded onto glass coverslip in 12-well plates and treated with OH-dDHL in a dose-dependent manner. Cells were incubated with Fluo-8 AM for 1 h at 37 °C in the dark and further fixed with 4% PFA. The coverslips were washed and mounted on microscope slides by using Vectashield mounting medium containing DAPI and stored at 4 °C in the dark. The stained samples were viewed with a Leica DMi8 confocal microscope (Leica Microsystems Ltd., Wetzlar, Germany).

The measurement of calcium influx into cells was adapted from Schaff et al (2008 (link)). After neutrophil isolation, the cells were resuspended in HBSS without calcium and magnesium (Lonza). Five micrometre Fluo‐8 AM (Abcam) was added to PMNs at 37°C for 90 min. Cells were washed once and resuspended in RPMI 1640. In a total reaction volume of 120 μl, 1 × 10⁵ cells were seeded into a black 96‐well plate and stimulated with 70, 15, 3 and 0.56 μM candidalysin. After 10 min incubation, the fluorescence was measured (Ex490/Em520) for 60 min with Fluostar Omega plate reader (BMG). Each experiment was performed in three technical replicates.

The level of intracellular Ca²⁺ was measured using Fluo 8-AM (ab142773, Abcam) according to the manufacturer’s instruction. In brief, prRGCs were seeded on 6-channel μ-Slide (80,606, ibidi) and cultured for 14 days. Cells in control group and EtOH group (5%, 3 h) were incubated with 4 μM Fluo 8-AM diluted in medium for 30 min at 37 °C. Cells were then washed 3 times with culture medium and imaged under FEI CorrSight microscopy with the laser line of 488 and the 446/523/600/677 nm BrightLine® quad-band bandpass filter. Fluorescence intensity was quantified with Fiji.

To observe ROS production in brain tissue of the mice from photothrombotic stroke and bEnd.3 cells, the tissue slices, and treated bEnd.3 cells were stained with 10 μM DCFH-DA (Beyotime, S0033S) and DAPI (Bioworld Technology, BD5010) at 37 °C for 20 min protected from light. Images of brain slices were viewed with the confocal laser-scanning microscope (Zeiss, LSM 800, Jena, Germany).
To observe the accumulation of mitochondrial ROS, the treated bEnd.3 cells were incubated with 5 μM MitoSOX™ red mitochondrial superoxide indicator (Invitrogen™, M36008, Carlsbad, CA, USA) reagent at 37 °C for 10 min, followed by 50 nM Mito-tracker green (Beyotime, C1048, Shanghai, China) reagent for 30 min in the dark. The nucleus was visualized by incubating with Hoechst (1:1000, Invitrogen™, H3570) for 10 min. Images were acquired and quantified by a confocal scanning microscope (Zeiss, LSM 800, Jena, Germany).
For the detection of intracellular calcium content, bEnd.3 cells with indicated treatment were incubated with Fluo-8AM (1 μg/mL, Abcam, ab142773, Cambridge, UK) for 30 min. After washing with PBS, the relative fluorescence intensity was detected using a multimode microplate reader (BERTHOLD Technologies, Bad Wildbad, Germany) at excitation of 490 nm and emission of 520 nm.