Fluo 3 am solution

Cells treated with zymosan and medium 199(Gibco, Grand Island, NE, USA) or melatonin (Sigma, St Louis, MO, USA) were incubatedin the presence of a 5µL Fluo-3 AM solution (Sigma, St Louis, MO, USA). The cells were washed (160 × g, 10 min, 4 °C) and resuspended in a HBSS (Hank’s Balanced Salt Solution) containing bovine serum albumin (BSA) and analyzed by Fluoroskan Ascent FL^®Microplate reader (Thermo Scientific, Vantaa, Finland), with the 485 nm excitation and 538 nm emission filters. The results were expressed as the mean fluorescence intensity of Fluo-3 AM. The experiments were performed in duplicate.

The cells were incubated with Zymosan in the presence of the 5-μL Fluo-3 AM solution (Sigma, St Louis, MO, USA). The cells were washed and resuspended in HBSS (Hank’s Balanced Salt Solution) containing bovine serum albumin (BSA). The fluorescence intensity was measured by a Fluoroskan Ascent FL^™ Microplate reader (Thermo Scientific, Vantaa, Finland) using 485-nm excitation and 538-nm emission filters. The rate of intracellular Ca²⁺ release was expressed as the mean fluorescence intensity of Fluo-3. The experiments were performed in duplicate.

Intracellular Ca²⁺ was detected as per previous study (Sun et al., 2015 (link)). For the in-vitro experiments, drug-treated neurons were rinsed three times with Hanks’ balanced salt solution (HBSS, Sigma-Aldrich, #H6648, St. Louis, MO, United States) and incubated with HBSS containing 10 μM Fluo-3-AM solution (Sigma-Aldrich, #39294, St. Louis, MO, United States) for 60 min at 37°C. For the in-vivo experiments for Ca²⁺ detection, tissue sections were first penetrated with HBSS containing 0.2% Triton X-100 and 0.1% sodium citrate (Solarbio Life Sciences, #C1032, Beijing, China) for 30 min, then incubated with HBSS containing 10 μM Fluo-3-AM solution for 60 min at 37°C. After incubation with Fluo-3-AM solution, the neurons and tissue sections were rinsed three times with HBSS. Observations were performed with a fluorescent microscope at a detection wavelength of 488 nm. Obtained images were analyzed with ImageJ.