Fluo 4 am dye

Animals were briefly anesthetized and euthanized with rapid decapitation. The brain was removed, and lateral ventricle and third ventricle CPs were harvested and placed in warm DMEM high glucose without phenol red. Suspended tissues were allowed to equilibrate at 37°C for 30 minutes. Subsequently, tissues were incubated in either Fluo-4 AM dye (Invitrogen, F14201) or CoroNa Green AM dye (Invitrogen, C36676) for 30 minutes at 37°C according to Invitrogen protocols. Tissues were washed twice with DMEM high glucose without phenol red. The TRPV4 agonist, ionomycin, or nystatin were added, and the tissues were imaged on a Keyence BZ-X800 epifluorescence scope. Image processing was done in the Keyence BZ-X800 Analyzer software, and figures were arranged in PowerPoint.

The intracellular Ca²⁺ levels in DU-145 and PC-3 cells were determined using flourimetry with the Fluo-4 AM dye (Invitrogen Carlsbad, CA, U.S.A.). Cells were cultured in specialized 96-well plates and loaded with 5 μM of Fluo-4 AM fluorescent dye for 30 min at 25°C. Experiments were performed in Hanks’ Balanced Salt Solution (HBSS) solution containing (mM); NaCl, 142; KCl, 5.6; MgCl₂, 1; CaCl₂, 2; Na₂HPO₄, 0.34; KH₂PO₄,0.44; HEPES, 10; glucose, 5.6; buffered to pH 7.4 with NaOH. The Ca²⁺ −free HBSS had the same constituents as HBSS solution, but with no CaCl₂ and with EGTA 1 mM added to eliminate any possible calcium contamination. Fluorescence measurements were performed at an excitation of 488 nm and an emission of 522 nm Fluostar Omega Spectrofluorimeter (BMG Technologies, Offenburg, Germany). All the experiments were repeated at least thrice.

A total of 25,000 cells were seeded into 35 mm glass bottom dishes. Next day, cells were incubated with 2 μM Fluo-4, AM dye (Invitrogen, F10489) in serum free medium for 30 min. Live cell imaging was performed using IX83 microscope (Olympus) with FITC filters at 37 °C in 5% CO₂. Niclosamide, inactive niclosamide analog or DMSO (0.1%) was added to the media 1 min after the start of live cell imaging. Events were recorded every 10 s. Quantitative analyses were performed using ImageJ. Ca²⁺ changes expressed as ΔF/F₀ values vs. time, F₀ is the background subtracted initial intensity value of the fluorescence. Background fluorescence was subtracted from each data point and was normalized using F₀ as a reference point. Finally, changes in average fluorescence intensity was graphically represented for each condition.

Hippocampal cells were removed from the plates and previously incubated with Fluo4-AM dye (Invitrogen) dissolved in Neurobasal medium (Gibco®) for one hour46 (link). The cells were centrifuged (300 g for 5 minutes at 20 °C), washed with phosphate buffer solution (PBS) without calcium, and plated in microscopy confocal (Zeiss LSM 510, Meta) microplates pretreated with poli-L-lysine. The fluorescence was measured for 90 seconds in the absence and/or in the presence of 10⁻⁵, 10⁻⁴ and 10⁻³ M AEME. After this period, 250 mM KCl was added in order to promote neuronal death and, thus, verify its viability. All measurements were performed with a PBS free-calcium solution. Digitalized images were analyzed through the software Origin 5.

3D gels with astrocytes (50–100 µL at 8 × 10⁶ astrocytes mL⁻¹) or 2D astrocytes (100 000 cells) cultured on MEAs were matured for 5 days prior to the recording, on the day of the recording—gently rinsed with PBS, and loaded with Fluo‐4 AM dye (5 µm, Invitrogen) and 0.04% pluronic F‐127 (Life Technologies) non‐ionic surfactant in Live Cell Imaging Solution (Invitrogen) for 1 h at 37 °C. The dye was then washed off, and cultures were allowed to acclimate for 1 h at 37 °C in the Neuronal Maintenance medium prior to re‐positioning into 35 mm µ‐dishes (re‐positioning step is applicable to 3D gels only) and recording with a spinning disk confocal microscope (Nikon) at 5 Hz sampling rate (0.2 s acquisition time per image) at 37 °C in a CO₂‐controlled chamber. At least three independent cultures were sampled per condition. ΔF/F₀ graphs were generated in Fiji software where cell bodies of interest were manually selected. Fluorescence intensity was measured at each cell body over time and normalized to its initial baseline fluorescence value.

NRCMs were cultured on confocal dishes and incubated with 3 μM fluo-4/AM dye (Invitrogen, Germany) for 30 min at 37 °C. Subsequently, the NRCMs were rinsed with Ca²⁺-free Tyrode’s solution (composed of 132 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl₂, 5 mM glucose, 10 mM HEPES, and 1.8 mM CaCl₂ at pH 7.4) and placed on the stage of a confocal laser scanning microscope (SP5-FCS, Leica). To induce transient intracellular Ca²⁺ release, the NRCMs were stimulated with either 1 μM or 10 μM adenosine 5’-triphosphate (ATP). Alternatively, to induce ER emptying, the NRCMs were stimulated with 2 μM thapsigargin (TG, Sigma-Aldrich). To investigate the role of IP3R in Ca²⁺ release, the NRCMs were pre-treated with 0.1 μM Xestospongin-C (XeC, Sigma-Aldrich) for 15 min before ATP stimulation. To analyze the calcium imaging results, the fluorescence change over time was quantified as F₁/F₀, where F₁ represents the maximal fluorescence, and F₀ represents basal fluorescence.