Fluo 4 acetoxymethyl am ester

Intracellular calcium levels were measured with the fluorescent calcium indicator, Fluo-4 acetoxymethyl (AM) ester (ThermoFisher Scientific, USA). Fluo-4 AM diluted in neuronal media was added to the neuronal cells and incubated for 30 min at 37°C in a humidified atmosphere (5% CO₂, 95% air). After 30 min incubation, dye solution was removed and cells were washed with PBS. Fluorescence was measured at 488 nm in FlexStation 3 (Molecular Devices, USA).

ES-2 and OV-90 cell lines were incubated on 6-well plates for 24 h in no-FBS medium. The cells were incubated with fucoidan (0, 25, 50, 100, 200, and 300 μg/mL) or co-treatment of fucoidan (300 μg/mL) and calcium chelators including 2-aminoethoxydiphenyl borate (2-APB; D9754, Sigma-Aldrich, St. Louis, MO, USA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester; BAPTA/AM; sc-202488, Santa Cruz Biotechnology, Dallas, TX, USA), or ruthenium red for 48 h at 37 °C and 5% CO₂. The cells were collected through 0.25% trypsin-EDTA and washed through medium before staining with 3 μM Fluo-4 acetoxymethyl (AM) ester (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5% CO₂ for 20 min. Fluorescent intensity was observed through Guava easyCyte™ 5 Flow Cytometer (Merck Millipore, Burlington, MA, USA). The experiment was repeated three times.

Relative intracellular calcium levels were assessed by using the calcium indicator Fluo-4 acetoxymethyl (AM) ester, which was obtained from Invitrogen Life Technologies (Grand Island, NY, USA). 1 μM Fluo-4 AM was loaded into cells by incubating cells at a concentration of 1 × 10⁶/mL for 30 min at 37 °C in loading buffer (20 mM HEPES, 20 mM Glucose and 1% BSA in PBS). After 10 min centrifugation at 400×g, cells were re-suspended in loading buffer and incubated further for 30 min at 37 °C until use, which allowed complete de-esterification of intracellular AM ester.

Calcium imaging was performed as previously described (Mutikainen et al., 2016 (link)). Cardiomyocytes were loaded with Fluo-4-acetoxymethyl (AM)-ester (2 μM, Invitrogen) in DMEM for 20 min in an incubator (37°C, 5% CO₂) and then coverslips with attached cells were placed into the recording chamber. Experiments were carried out after a period of 20 min to allow deesterification of the dye. [Ca²⁺]_i measurement was performed with a confocal inverted microscope (FluoView 1000; Olympus, Japan). To measure myocyte calcium [Ca²⁺]_i transients, the cells were excited at 488 nm and the emitted light (500–600 nm) was collected through water immersion 60X objective lens, using the line-scan mode. To stimulate the cells, myocytes were stimulated with 1-ms voltage square pulses (Grass stimulator, S48) 50% over the excitation threshold through platinum electrodes. In some experiments, caffeine (10 mM, Sigma) was applied directly to the studied area with a local perfusion manifold (Cell MicroControls, USA). Fluo-4 fluorescence intensity is expressed as an F/F₀-ratio, where F is the background subtracted fluorescence intensity and F₀ is the background subtracted minimum fluorescence value measured from each cell at rest. The images were analyzed with FluoView and ImageJ (imagej.nih.gov/ij/) softwares.

The relative cytosolic calcium levels were estimated using the sensitive dye Fluo-4 acetoxymethyl (AM) ester (Invitrogen Life Technologies, Grand Island, NY, USA). 1 μmol/L Fluo-4 AM was loaded into neutrophils by incubating and gently vibrating cells (10⁶/mL) for 30 min at 37°C in loading buffer (20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20 mmol/L glucose, and 1% BSA in PBS). After 10 min centrifugation at 400 ×g, neutrophils were re-suspended in loading buffer without dye, and, for complete de-esterification of intracellular AM ester, incubated for a further 30 min at 37°C until use.
To disrupt lipid rafts on membrane, depolymerize the cytoskeleton actin, or block IP3, the membrane calcium channel, Syk, and moesin, neutrophils were preincubated with lipid raft disruptor MβCD (5 mmol/L) and cytochalasin B (5 μg/mL) for 5 min, or IP3 inhibitor 2-APB (100 μmol/L) for 8 min, membrane calcium channel inhibitor LaCl₃ (10 μmol/L) for 30 min, Syk inhibitor piceatannol (20 μmol/L) for 30 min, moesin inhibitor staurosporine (0.01 μmol/L) for 30 min, or 0.1% of DMSO as vehicle control.

Calcium signaling was analyzed in 3D SG models cultured with primary facial or axillary SG cells. Experiments were performed in 96-well black-walled, clear-bottom microplates (#CLS3340, Sigma-Aldrich) and in indicator-free DMEM media (#31053, Gibco) at RT. Cells were incubated with 4 μM fluo-4 acetoxy-methyl (AM) ester (#F14217, Invitrogen) for 1 h after washing the cultures twice with PBS. De-esterification was done for 1–2 h to allow cleavage of the fluorophores by intracellular esterases, following the manufacturer’s protocol. 2,5 mM Probenecid (#P8761, Sigma-Aldrich) was added to the media preventing the efflux of the calcium indicator after loading. After baseline recording (excitation 485 nm, emission 535 nm), cells were stimulated with agonists ACh, pilocarpine, carbachol, adenosine triphosphate (ATP), and/or inhibited with antagonists glycopyrrolate (all Sigma-Aldrich). Relative change for intracellular calcium fluxes was calculated by equation ΔF = (F_max−F0)/F0 in at least three independent experiments with n = 6±SEM.

Dulbecco’s modified Eagle’s medium (DMEM), hygromycin-B and Fluo-4 acetoxymethyl (AM) ester were purchased from Invitrogen (Carlsbad, CA, USA). Foetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Melbourne, VIC, Australia). The QuikChange™ site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA, USA). AlphaScreen™ reagents, Bolton-Hunter reagent [¹²⁵I] and 384-well ProxiPlates were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA). SureFire™ ERK1/2 reagents were generously supplied by TGR Biosciences (Adelaide, SA, Australia). SigmaFast o-phenylenediamine dihydrochloride (OPD) tablets and antibodies were purchased from Sigma–Aldrich (St. Louis, MO, USA). GLP-1 peptides were purchased from Mimotopes (Clayton, VIC, Australia). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) or BDH Merck (Melbourne, VIC, Australia) and were of an analytical grade.