Cytosolic Ca2+ and pH were measured independently using the fluorescent ratiometric indicators Fura-2 and BCECF, respectively. All experiments were performed in HEPES-buffered saline (HBS) comprising 1.25 mM KH2PO4, 2 mM CaCl2, 2 mM MgSO4, 3 mM KCl, 156 mM NaCl, 10 mM glucose and 10 mM HEPES (pH 7.4; all from Sigma-Aldrich). For measurement of Ca2+, cells were incubated with Fura-2 AM (2.5 µM) and 0.005% (v/v) pluronic acid (from Invitrogen) for 1 h in HBS. Fura-2 was excited at 340/380 nm and emitted fluorescence was captured using a 440 nm long pass filter and a 20× objective. For the measurement of pH, cells were incubated with BCECF-AM (5 µM) and 0.005% v/v pluronic acid (Invitrogen) for 30 min in HBS. BCECF was excited at 490/440 nm and emitted fluorescence was captured using a 515 nm long pass filter and a 20× objective.
Pluronic acid
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy, Germany
Pluronic acid is a co-polymer composed of polyethylene glycol (PEG) and polypropylene glycol (PPG) blocks. It is a nonionic surfactant commonly used in various applications, including pharmaceutical, biomedical, and industrial formulations. Pluronic acid has the ability to modify the physical and chemical properties of solutions, making it a versatile component in various laboratory and industrial processes.
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Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (45 mentions)
Fluo 4 am, by Thermo Fisher Scientific (34 mentions)
Fluo 3 am, by Thermo Fisher Scientific (7 mentions)
Lsm 700, by Zeiss (6 mentions)
Probenecid, by Merck Group (6 mentions)
Fm4 64, by Thermo Fisher Scientific (5 mentions)
Tyrode s solution, by Merck Group (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Fluo 4, by Thermo Fisher Scientific (3 mentions)
Ix83xdc, by Olympus (3 mentions)
Corona green am, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Ionomycin, by Alomone (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Culturewell, by Grace Bio-Labs (3 mentions)
Lsm 510 confocal system, by Zeiss (2 mentions)
Climate box, by Pecon (2 mentions)
116 protocols using pluronic acid
1
Simultaneous Measurement of Cytosolic Ca2+ and pH
2
Intracellular Calcium Flux Visualization
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Intracellular Ca++ fluxes were visualized by the Ca++-sensitive dye fluo-4 AM (Invitrogen) following the manufacturer’s recommendations. Briefly, host cells were incubated for 30 min in fluo-4 AM (2.5-μM final concentration) at 37 °C and supplemented with pluronic acid (Invitrogen) in a dye/pluronic acid ratio of 1:1. Non-incorporated dye was removed by washing in sterile PBS and adding fresh modECGM. Calcium influx induction was induced by calcium ionophore A23187 treatments (15 μM, Sigma-Aldrich).
Post-processing analysis was performed by the use of Image J v1.52p software (Schneider et al. 2012 (link)). Z Project plugin was applied to holotomographic z-stacks with the average intensity of the images as projection output. For calcium flux measurements over time, defined areas surrounding resting host cells were defined as regions of interest (ROI) (Silvestre-Roig et al. 2019 (link)). Thereafter, the multi-measurement tool (roiManager “Multi Measure”) was used to quantify the mean grey value as indicator of fluorescence intensity over time. Finally, for better visualization of Ca++ flux, fluorescence images were displayed in pseudo-colours, using fire lookup tables as described elsewhere (Ardiel et al. 2017 (link); Liu and Baraban 2019 (link); Wakida et al. 2020 (link)).
Post-processing analysis was performed by the use of Image J v1.52p software (Schneider et al. 2012 (link)). Z Project plugin was applied to holotomographic z-stacks with the average intensity of the images as projection output. For calcium flux measurements over time, defined areas surrounding resting host cells were defined as regions of interest (ROI) (Silvestre-Roig et al. 2019 (link)). Thereafter, the multi-measurement tool (roiManager “Multi Measure”) was used to quantify the mean grey value as indicator of fluorescence intensity over time. Finally, for better visualization of Ca++ flux, fluorescence images were displayed in pseudo-colours, using fire lookup tables as described elsewhere (Ardiel et al. 2017 (link); Liu and Baraban 2019 (link); Wakida et al. 2020 (link)).
3
Visualizing Cardiomyocyte Calcium Sparks
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Myocytes on laminin‐coated recording chambers were loaded with 10 μmol/L Fluo‐4 acetoxymethyl ester in the presence of 0.02% (w/v) pluronic acid (Molecular Probes; 15 min incubation), mounted on stage of a laser‐scanning confocal microscope (LSM 7, Zeiss), and superfused with normal Tyrode solution. Ca sparks were assessed at room temperature as described previously.19 For some experiments, CaMKII was inhibited with RA608 at concentrations of 1, 3, or 10 μM. For comparison, the CaMKII inhibitor AIP (in its myristoylated form) was also investigated in some experiments (2 μM).
4
Intracellular Calcium Dynamics Imaging
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The levels of intracellular Ca2+ were determined using an LSM 510 confocal system from Carl Zeiss through computer-assisted video imaging on a single-cell basis. SH-SY5Y cells were treated onto 25 mm coverslips according to the experimental protocol. Following that, the cells were loaded with 4 µM Fluo-4/AM (Molecular Probe, Eugene, OR, USA) dissolved in Ca-PSS supplemented with 0.08% pluronic acid (Molecular Probe) for 40 min in darkness at RT. Cells were rinsed once with Na-PSS and subsequently treated with 1 μM thapsigargin in Na-PSS for 10 min [40 (link)]. By switching Na-PSS to K-PSS, we assessed the uptake of Ca2+ via the reverse mode. A peristaltic pump was used to change bath solutions, and images were captured every 5 s. A heated microscope stage and climate box from PeCon GmbH were employed to maintain the cells and perfusion solutions at 37 °C. An argon laser at 488 nm provided the excitation light, and the emission in the range of 505–530 nm was recorded in a time-lapse manner. Following image acquisition, we conducted the fluorescence intensity analysis offline, following the previously described methodology [40 (link)].
5
Fura-2 Calcium Imaging on Cultured Cells
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Cells were plated on collagen/poly-D -lysine glass coverslips, loaded with Fura-2 acetoxymethyl ester (2.5–5 mM), and incubated for 60 min at room temperature in 1.5 mM of pluronic acid (Molecular Probes, Eugene, OR) in a HEPES-buffered saline (2 mM Ca2+). Coverslips were placed in a laminar flow perfusion chamber (Warner Instrument Corp.) and constantly perfused with HEPES-buffered saline (2 mM Ca2+). Images of Fura-2-loaded cells with the excitation wavelength alternating between 340 and 380 nm were captured. Following subtraction of background fluorescence, the ratio of fluorescence intensity at the two wavelengths was calculated. Ratio levels were analyzed using MetaFluor (Universal Imaging Corporation).
6
Neuronal Culture Reagents and Protocols
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Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium (MEM), deoxyribonuclease (DNase), poly-L-lysine, poly-D-lysine hydrobromide, cytosine arabinoside, NMDA, protease inhibitor cocktail, MK-801, and ifenprodil were purchased from Sigma (St. Louis, MO, USA). Pluronic acid and Fluo-3 AM were purchased from Molecular Probes (Eugene, OR, USA). TCN-201, IPA-3, and NSC23766 were purchased from Tocris (Bristol, UK). Pierce ECL kits (Thermo Fisher Scientific, Rockford, IL, USA), Neurobasal, and B-27 supplements were purchased from Invitrogen Corporation (Carlsbad, CA, USA); the p-PAK1 antibody (Thr 212) from Santa Cruz Biotechnology (Dallas, TX, USA); and the PAK1 antibody anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA). Brevetoxin-2 (PbTx-2) was isolated and purified from Karinia breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC, USA). QNZ-46 was a gift from SF Traynelis, Department of Pharmacology, Emory University, Atlanta, GA. The GluN2D subtype of NMDA receptor knockout mice was obtained from Daniel T. Monaghan, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska.
7
Mitochondrial Free Zinc Dynamics
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The free zinc (Zn2+) concentration in the mitochondria of H9c2 cells was assessed with RhodZin-3 according to the manufacturer's instructions (MolecularProbe) and previous studies [29 (link), 30 (link)]. After delayed preconditioning induced by diazoxide or 48 hours after siRNA transfection, H9c2 cells were cultured in a specific temperature-controlled culture dish. The dish was coloaded with both 5 μM RhodZin-3 plus 0.02% pluronic acid (MolecularProbe) and 100 nM Mito-Tracker Green (Beyotime, China) in fresh medium without FBS for 30 min at 37°C in the dark in a humidified 5% CO2 + 95% air atmosphere. After 3 washes with phosphate-buffered saline, the cells were incubated for another 30 min for deesterification and then mounted on the stage of confocal microscope in a humidified 5% CO2 + 95% air atmosphere (Leica, Germany). The temperature was maintained at 37°C with a Delta T Open Dish System (Bioptechs, Butler, PA), and the fluorescence intensity was measured as described above with Image Pro-Plus 5.1.
8
Monitoring Astrocyte Cytosolic Calcium
9
Calcium Flux Monitoring in Activated T Cells
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T cells were loaded with 3μM each of Fluo4 and Fura red (Molecular Probes, Invitrogen) for 45 minutes in the dark at 37°C in cell loading medium (DMEM medium with 10mM HEPES and 10% fetal calf serum) in the presence of 0.01% pluronic acid (Molecular Probes). Dye-loaded cells were rinsed twice with loading medium, incubated with 10μg/ml mouse anti-CD3 (BD Biosciences) and 2.5μg/ml mouse anti-CD28 (BD Biosciences) for 30 minutes at room temperature, and then rinsed again with loading medium to be ready for calcium flux studies. Cells were acquired with FACS LSRII and cell activation was initiated by addition of goat anti-mouse antibody (Jackson ImmunoResearch) to final 10μg/ml. CD4 T cells were also stimulated with 1μM thapsigargin. Kinetics of calcium flux in activated cells were monitored by reading the calcium dependent Fluo4 emission signals at 530nm compared to non-specific Fura red signals at 685nm. Calcium flux was calculated as ratio of Fluo4 to Fura red emission signals.
10
Quantifying Cellular Oxidative and Nitric Oxide Levels
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Chloromethyl-dichloro-dihydro-fluorescein diacetate (DCFDA, Molecular Probes, Life Technologies, USA) was used for ROS detection, and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM, Molecular Probes, Life Technologies, USA) for NO detection as fluorescent probes, as described in our previous studies [39 (link), 40 (link)]. Brain endothelial cells were cultured in 96-well plates with black walls and transparent plastic bottoms (Corning, NY, USA). After treatments for 1 or 24 h, cells were incubated in Ringer–Hepes buffer containing 2 µM DCFDA or 2 µM DAF-FM probes. Pluronic acid (Molecular Probes, Life Technologies, USA; 16 µM) was used to help the probes crossing the cell membrane. Fluorescence was detected by Fluostar Optima multiwell plate reader (BMG Labtechnologies, Germany) at 485 nm excitation and 538 nm emission wavelengths at every 3 min for 1 h.
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