Caco-2 cells were seeded on 8-well chambered coverglass (Nunc Lab-Tek, Thermo Fisher, MA, USA). Cells were washed with Hanks’ balanced salt solution containing 10 mM HEPES, 0.1 mM CaCl2 and 1 mM MgCl2, and then loaded with a mixture of 4 μM Fluo-4 acetoxymethyl (Fluo-4 AM; Thermo Fisher) and 20% pluronic acid (Sigma) for 45 min at 37 °C. The solution was discarded, and buffer was added into the chamber for 30 min to ensure complete de-esterification of AM ester. Loaded cells were mounted under a CLSM for live-cell imaging and were sequentially incubated for 3 min each with buffer (baseline), 100 nM PTH, and 5 μM ionomycin (positive control). Images were acquired every 5 s and quantified for fluorescence intensities.
Pluronic acid
Manufactured by Merck Group
Sourced in United States, Germany
Pluronic acid, also known as poloxamer, is a non-ionic triblock copolymer composed of a central hydrophobic block of polypropylene glycol (PPG) flanked by two hydrophilic blocks of polyethylene glycol (PEG). It is a versatile and widely used laboratory compound due to its unique physical and chemical properties.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (4 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Fura 2 am, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Potassium chloride (kcl), by Merck Group (3 mentions)
Amphotericin b, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
F1141 2mg, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Bradykinin, by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Merck Group (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Tunicamycin, by Merck Group (2 mentions)
Cacl2, by Merck Group (2 mentions)
4 chamber 35 mm glass bottom petri dish, by Cellvis (2 mentions)
Streptomycin, by Merck Group (2 mentions)
29 protocols using pluronic acid
1
Calcium Imaging of Caco-2 Cells
2
Fibronectin Micropatterning for Cell Adhesion
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Fibronectin micropatterning was carried out. Polydimethylsiloxane (PDMS) elastomer (SYLGARD 184; Dow Corning) was used in a 1:10 ratio of curative-to-precursor according to the manufacturer’s protocol. This mixture was poured into microfabricated silicon wafers. These wafers were degassed for 45 min. To prepare stamps, the wafers were kept in the oven at 80 °C for 3 h. Freshly made PDMS stamps were oxidized and sterilized under high power in Plasma Cleaner (Model PDC-002; Harrick Scientific) for 5 min. 10% fibronectin solution was allowed to adsorb onto the surface of each PDMS stamp under sterile condition. The PDMS stamp was then deposited onto the surface of hydrophobic dishes (uncoated dish from ibidi) to allow transferring of the micro features. The surface was then treated with 2 mg/mL Pluronic F-127 (Sigma) for 30 min to passivate non–fibronectin-coated regions. Rectangles with an area of ~ 3300 μm2 (width 29 µm and length 116 µm) were stamped on uncoated Ibidi dishes. These micropatterned dishes were then passivated with 0.2% pluronic acid (Sigma P2443) for 5 min and washed twice with 1× PBS before cell seeding.
3
Worm Exposure to Barbadin
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Worms were added to the center of the test plate in an ∼10-μL drop of 1 mM Barbadin (Toronto Research Chemicals) (or DMSO in control conditions) dissolved in 2% pluronic acid (F-127, Sigma Aldrich) in M9. Barbadin was prepared as a stock solution at 100 mM in DMSO. In addition to Barbadin in the drop at the center of the plate, the agar in these experiments contained 100 µM Barbadin (or DMSO in control conditions) and 2% pluronic acid.
4
Fluo-4 Ca2+ Indicator Loading in Myocytes
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Cells were loaded with the AM form of the Ca2+ indicator Fluo‐4, Fluo4‐AM (Molecular Probes, Thermo Fisher Scientific, F‐14201) to provide a wide range of sensitivity to changes of [Ca2+]i. Fluo‐4 powder was dissolved in DMSO containing 25% Pluronic acid (Sigma‐Aldrich) at a concentration of 1 μg μl−1. Myocytes were then incubated with Fluo‐4 Ca2+ indicator solution for 15 min followed by 45 min in normal Tyrode solution to permit the Ca2+ indicator to de‐esterify. Measured fluorescence was normalized to resting levels (F/F0).
5
In-situ pH Calibration for Plant Imaging
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Staining protocol as described by (Bassil et al., 2013 ) was adopted. pH determinations were based on a calibration curve generated with buffers prepared for pH 5.8–7.4 with increments of 0.4 pH units. pH calibration solutions were made in 50 mM ammonium acetate and 50 mM 2‐morpholinoethanesulfonic acid (MES) (for pH standards 5.8–6.2) or 50 mM (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) (HEPES) (for pH standards 6.6–7.4). Required pH values were obtained by adjustment using 50 mM Bis‐tris propane (BTP) (Sigma, MO, USA). 2′,7′‐Bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, acetoxymethyl ester (BCECF‐AM) (1.6 mM stock) was used with a working concentration of 10 μM along with 0.02% pluronic acid (Sigma). BCECF‐AM preloaded 5‐day‐old wild‐type plants were incubated in respective pH standard solutions containing 10 μM nigericin and valinomycin (Sigma, MO, USA) for 15 min and then imaged at 20x magnification, following sequential excitation at 445 and 488 nm and emission recorded at 510–550 nm. Fluorescence intensity ratios were determined, and pH was plotted as log regression to generate an in‐situ calibration curve, used to determine pH values for experimental plants.
6
Fibronectin Micropatterning for Cell Studies
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Fibronectin micropatterning was performed as described previously (23 (link)). Briefly, 1,800 μm2 rectangles (aspect ratio 1:5) (RE), 1,800 μm2 circles (BC), and 500 μm2 circles fibronectin (Sigma F1141-2MG) micropatterns (area = 1,800 μm2 and aspect ratio = 1:5) were made on uncoated Ibidi dishes (81151). These micropatterned dishes were then passivated with 0.2% pluronic acid (Sigma P2443) for 10 min and washed with PBS.
7
Calcium Imaging of Neuronal Cells
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After 24 h of incubation, the cells were loaded with Hank’s Balanced Salt Solution (HBSS) supplemented with 2.5 mM probenecid (Sigma-Aldrich, Milan, Italy), 3 μM of the calcium sensitive fluorescent dye Fluo-4 AM (Thermo-Fisher Scientific, Waltham, MA, United States), 0.01% pluronic acid and 20 mM HEPES (pH 7.4; Sigma-Aldrich, Milan, Italy) for 30 min at 37°C. Afterwards the loading solution was aspirated, a washing step with 100 μl/well of HBSS, HEPES (20 mM, pH 7.4), 2.5 mM probenecid and 500 μM Brilliant Black (Sigma-Aldrich, Milan, Italy) was carried out. Subsequently 100 μl/well of the same buffer were added for 10 min. Concentrated solutions of ligands (i.e., glutamate, L -quisqualate, CHPG and GET73) were freshly prepared and serial dilutions were made in HBSS/HEPES (20 mM) buffer (containing 0.02% BSA fraction V). After placing cell culture and compound plates into the FlexStation II (Molecular Devices, Sunnyvale, CA, United States), the on-line additions were carried out in a volume of 50 μl /well and fluorescence changes were continuously measured for 2 min at 37°C. The calcium peak level has been then selected to evaluate the effects of treatments.
8
Fibronectin Micropatterning for Cell Adhesion
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Fibronectin micropatterning was performed as described by Makhija et al [50 (link)]. Briefly, circular fibronectin (Sigma F1141-2MG) micropatterns (area = 1800μm2) were made on uncoated Ibidi dishes (81151). These micropatterned dishes were then passivated with 0.2% pluronic acid (Sigma P2443) for 10 minutes and washed with PBS.
9
Fluorescent Nucleic Acid Staining Protocol
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Fluorescent nucleic acid stain Hoechst 33258 was purchased from Molecular Probes (Eugene, OR, USA). Thapsigargin,Tunicamycin, EGF, Rapamycin, Bafilomycin A, Fura 2AM, NaCl, KCl, pluronic acid, CaCl2, MgCl2, glucose, HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-acetyl-Asp-Glu-Val-Asp-7 amido-4-methylcoumarin and Bradykinin were purchased from Sigma Aldrich (Munich, Germany). Suc-Leu-Leu-Val-Tyr-AMC was received from Enzo (Lausen, Switzerland). Alamar Blue was purchased from Invitrogen, Thermo Fischer Scientific, Carlsbad, CA, USA. The CYTO-ID Autophagy detection kit was purchased from Enzo.
10
Quantifying Calcium Dynamics in Activated Neutrophils
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Calcium analysis was performed using flow cytometry (C6 Accuri, BD). Human primary neutrophils were stimulated with LPS (100 ng/ml) for 3 h, washed and incubated with pluronic acid and fluo-3-acetoxymethyl ester (10 µM; Sigma-Aldrich, France) for 30 min in Ringer’s solution (145 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, and 0.1% bovine serum albumin, pH adjusted to 7.5). After baseline acquisition for 1 min, intracellular calcium mobilization was triggered by stimulation with the TREM-1 agonist, and the cells were monitored for 3 min. The results were normalized between the baseline and the maximal signal induced by ionomycin during the last minute of acquisition (1 µM; Sigma-Aldrich, France).
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