Fluo 2am

Cells overexpressing or depleted of GPT2 were incubated in Hank's balanced salt solution containing the calcium-sensitive fluorescent dye Fluo-2/AM (Cat# F1201, Invitrogen, CA, USA) for 30 min at 37 °C, and picrotoxin or CGP52432 were added at concentrations of 100 μM and 33 μM, respectively. Fluorescence intensity was measured with an Olympus confocal laser scanning microscope (DU-897D-CS0) and MetaMorph software, where excitation was performed at 340 and 380 nm, after which a comparative analysis of the two emission values was performed. Serial scanning was performed at excitation wavelengths of 340 and 380 nm at 2 s intervals, and the fluorescence intensity of each emission from each cell was detected. The fluorescence intensity changes (F340/F380) indicate the intracellular calcium concentration.

Cultures were initially incubated with Fluo-2AM (2 μM, Invitrogen, ThermoFisher Scientific, Inc., Waltham, MA, USA) for 1 h at room temperature to image variations in [Ca²⁺]c resulting from network activity. Cultures were then placed in the 3 mL perfusion chamber, on the stage of an upright Zeiss 880 confocal microscope using a 40× water immersion objective (1.0 NA), and imaged at a rate of 10–20 frames/s. No photobleaching was detected under these conditions. Standard medium for imaging contained (in mM) NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10, pH was adjusted to 7.4 with NaOH and osmolality to 320 mOsm with sucrose. Imaging of cell morphology (405 nm), cytosolic (488 nm) and MT calcium (543 nm) were made simultaneously. All measurements were conducted with identical laser parameters for all groups (e.g., intensity, optical section, duration of exposure and spatial resolution) at room temperature. Pharmacological agents were applied into the chamber during recording. The substances were dissolved at working concentration and the initial volume was gradually replaced with this solution during 30–60 s. The substances were considered to be completely washed in or out the chamber after replacing the entire volume of the chamber two-three times.

[Ca²⁺]_i measurements were taken to assess ALX/FPR2 receptor activity following stimulation with the following agonists: Carbachol (Cch; 100 μM), AT-RvD1 (100 ng/mL), as well as PBS (negative control). First, submandibular glands (SMGs) from C57BL/6, NOD/ShiLtJ, and ALX/FPR2 knockout mice were isolated and dissociated using a GentleMACS Tissue Dissociator (Miltenyi Biotec Inc., Sand Diego, CA), then plated on Cell-Tak (BD Biosciences, San Jose, CA) in 8-well chambers mounted on German borosilicate coverglasses (Nalge Nunc International, Penfield, NY). Cells were allowed to adhere to Cell-Tak for 30 mins, then were pre-loaded with fluo 2-AM (Invitrogen, Carlsbad, CA) for 20 min at 37 °C. Cells were washed three times with DMEM/F-12. [Ca²⁺]_i release was measured using a fluorescence microscope (Leica Microsystems) and time-lapse settings. Cch, AT-RvD1, and PBS were added to wells and cells were observed for changes in fluorescent intensity. Graphs plotting mean fluorescent intensity versus time were created using LAS (Leica Microsystems), Excel, and Graphpad Prism software.

Cultures were placed in the imaging chamber, on the stage of an upright Zeiss 880 confocal microscope using a 40x water immersion objective (1.0 NA), and imaged at a rate of 10-20 frames/s. No photobleaching was detected under these conditions. Experiments were also conducted on an inverted Zeiss 510 confocal microscope, in specific experiments. Standard recording medium contained (in mM) NaCl 129, KCl 4, MgCl₂ 1, CaCl₂ 2, glucose 10, and HEPES 10, pH was adjusted to 7.4 with NaOH and osmolality to 320 mOsm with sucrose. Cultures were incubated with Fluo-2AM (2 μM, Invitrogen) for 1 hour at room temperature to image variations in [Ca²⁺]i resulting from network activity or changes in ambient [Ca²⁺]o. Imaging of cell morphology (401 nm), calcium variations (488 nm), and mitochondrial calcium (550 nm) was made with the appropriate wavelengths. All measurements were conducted with identical laser parameters for all groups (e.g., intensity, optical section, duration of exposure, and spatial resolution).

The cardiomyocytes were seeded into 6-well culture plates (3 × 10⁵ cells/well) and then exposed to Fluo-2/AM (Molecular Probes, USA) for 30–40 min at 37°C in the dark, then transferred into a chamber placed on an inverted Nikon Diaphot microscope (Diaphot; Nikon; Tokyo, Japan). Next, illuminated the formulation with the exciting light of a 488 nm light and 488 nm (model D104, Photon Technology International, Inc., South Brunswick, NJ, USA). A photon-counter was used to identify the fluorescence signal and variations. The fluorescence measurement of Ca²⁺ was determined and verified at 10 Hz (Photon Technology). Variations in the emission fluorescence intensity were taken as changes in Ca²⁺.

Ca²⁺ transients were measured in isolated cultured DRG neurons incubated with 5 mM Fluo-2-AM (Molecular Probes, Invitrogen) for 20 min at 37 °C, and DRG were mounted in an open chamber and superfused with bath solution. The extracellular standard bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose at pH 7.4, adjusted with NaOH. Cytosolic free Ca²⁺ concentrations were measured by dual-wavelength Fura-2 microfluorometry with excitation at 340/380 nm and emission at 510 nm. Fura-2 fluorescence was recorded with a CCD camera, CoolSnap ES (Roper Scientific/Photometrics). Data were acquired using imaging processing software IPlab (Solution Systems, Funabashi, Japan) and analyzed with ImageJ 1.53 (NIH). At the end of each experiment, ionomycin (5 μM) was applied in the presence of 20 mM extracellular CaCl₂ to obtain saturating levels of Ca²⁺ influx as F_max. The population that did not respond to either molecular stimulus, responded to AITC alone, capsaicin alone, and the population responding to both stimuli were determined by the number of neurons responding to capsaicin and/or AITC divided by the number of neurons responding to ionomycin and expressed as a percentage.