Cryopreserved hESC-CMs were thawed, re-plated, and cultured for an additional 4 days for in vitro electrophysiological analysis. To examine the autonomic beating rate, the maximum dV/dt of depolarisation (dV/dtmax), and action potential duration at 50% and 90% repolarisation (APD50, and APD90, respectively), of spontaneous action potentials were recorded using a ruptured whole-cell patch-clamp technique in the current-clamp mode at 35–36 °C using a patch-clamp amplifier (Axopatch 200B, Molecular Devices) and sampled at 5 kHz after being low-pass-filtered at 2 kHz33 (link). Patch pipettes (7–8 MΩ) were fabricated from borosilicate glass capillaries (Kimax-51, Kimble Glass) and coated with Sylgard 184 (Dow Corning Toray Co.). Series resistance was always kept below 20 MΩ. Action potentials were measured using an intracellular solution containing (mmol/L): 130 potassium gluconate (Wako), 10 KCl (Wako), 5 NaCl (Wako), 1 MgCl2 (Wako), 0.1 EGTA (Dojindo), 0.1 Mg ATP, and 10 HEPES (Dojindo) [pH 7.2 with KOH (Wako)]. The extracellular bath solution contained (mmol/L): 136.5 NaCl, 5.4 KCl, 1.8 CaCl2 (Wako), 0.53 MgCl2, 5.5 HEPES, and 5.5 glucose (Wako) (pH 7.4 with NaOH).
Mgcl2
Manufactured by Fujifilm
Sourced in Japan
MgCl2 is a chemical compound commonly used in various laboratory applications. It is a white, crystalline solid with the chemical formula MgCl2. MgCl2 is an important source of magnesium and chloride ions, which are essential for many scientific and industrial processes.
Automatically generated - may contain errors
Lab products found in correlation
Cacl2, by Fujifilm (8 mentions)
Potassium chloride (kcl), by Fujifilm (5 mentions)
Sodium chloride (nacl), by Fujifilm (5 mentions)
Glucose, by Fujifilm (5 mentions)
N n dimethylformamide (dmf), by Fujifilm (3 mentions)
Naphthol as mx phosphate, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (2 mentions)
Zncl2, by Fujifilm (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fecl3, by Fujifilm (2 mentions)
Sucrose, by Fujifilm (2 mentions)
Hepes, by Dojindo Laboratories (2 mentions)
Fast blue bb salt, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Merck Group (1 mentions)
Cacl2, by Nacalai Tesque (1 mentions)
Dulbecco s phosphate buffered saline d pbs, by Nacalai Tesque (1 mentions)
Sodium chloride (nacl), by Junsei (1 mentions)
5 fluoresceinamine, by Merck Group (1 mentions)
Disodium p nitrophenyl phosphate, by Fujifilm (1 mentions)
25 protocols using mgcl2
1
Electrophysiological Characterization of Cryopreserved hESC-CMs
2
Alkaline Phosphatase Activity Assay
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Cell layers were rinsed with PBS in triplicate and then lysated with lysis buffer containing 0.5% Triton, 50 mM of Tris–HCl, and 5 mM of MgCl2 (Sigma). ALP activity was assayed at 37°C in a buffer containing 0.1 M 2-amino-2-methyl-1-propanol (Wako), 2 mM MgCl2, and p-nitrophenyl phosphate (pNPP) for 30 min. The reaction was terminated by adding of 200 ml of 2 M NaOH per 200 μl of reaction mixture. Absorbance values were measured at 405 nm using pNPP (Sigma-Aldrich) as the substrate.
3
Synthesis of Fluorescent Hyaluronic Acid Conjugates
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Sodium hyaluronate (Biohyalo 12, weight-average molecular weight 1100–1600 kDa) was obtained from SHISEIDO Co., Ltd. (Tokyo, Japan). MgCl2 and KCl were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). CaCl2, AlCl3, Dulbecco’s phosphate buffered saline (D-PBS) (−) were purchased from NACALAI TESQUE, INC. (Kyoto, Japan). NaCl was purchased from JUNSEI CHEMICAL Co., Ltd. (Tokyo, Japan). 5-fluoresceinamine was purchased from Sigma (St. Louis, MO, USA). 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinlum chloride (DMT-MM) was purchased from Kokusan Chemical Co., Ltd. (Tokyo, Japan). Deionized water was used as the solvent in all experiments unless otherwise stated.
4
Quantitative Assay for Alkaline Phosphatase
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Cells were cultured in the same conditions as described in the ALP activity staining subsection above, then washed with PBS and homogenized with 1% Nonidet P-40 (50 μL) under sonication on ice. Cell lysates (10 μL) were added to 50 μL of 0.2 mol/L Tris–HCl buffer (pH 9.5) containing 1 mmol/L MgCl2 and 12.5 mmol/L disodium p-nitrophenyl phosphate (Wako Pure Chemical Industries). After incubation for 15 minutes at 37 °C, reactions were terminated by addition of 50 μL of 0.5 mol/L NaOH and absorbance of the reaction mixture at 405 nm was read using a micro-plate reader (SH-1000, Corona Electric, Ibaraki, Japan). The increase in absorbance in after 15 minutes was divided by the amount of cellular protein and the obtained value was used to express the specific activity of ALP.
5
Osteogenic Differentiation of ADMPCs
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To induce osteogenic differentiation, ADMPC were cultured in 48 well plate until confluent and were then treated with osteogenic differentiation medium (ODM) consisting of MF medium supplemented with, 10 mM b-glycerophosphate, 50 µg/ml ascorbic acid, 10 nM dexamethasone, and 200 ng/ml recombinant human bone morphogenetic protein (rhBMP)-2 (R&D systems), as described previously45 (link). ADMPC without ODM served as a control group. Extracellular matrix calcification was determined on fixed ADMPC stained with 1% alizarin red solution (Wako) for five mins. ALP activity was detected by staining with ALP staining solution consisting of 0.1 mg/ml naphthol AS-MX phosphate (Sigma Aldrich), 0.6 mg/ml Fast Blue B salt (Sigma Aldrich), 2 mmol/L MgCl2 (Wako), 0.5% N,N dimethylformamide (Wako) and Tris-HCl (pH 8.5) at room temperature as described previously46 (link). Photographs were obtained to compare the intensity of staining between the control and ODM cultures.
6
Luciferase Assay for Gene Expression Analysis in Mice
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A luciferase assay was carried out as described previously (28 ). Briefly, female BALB/c mice were subjected to HD with 2.0 ml of PBS-containing 1.56 µg pCADEST1-Luc expressing the firefly luciferase gene (28 ) and 6.25 µg pCADEST1-anti-RP105 kappa-F2A-anti-RP105 mIgG1-TM or pCADEST1-anti-OVA kappa-F2A-anti-OVA mIgG1-TM. One day later, their livers were obtained, cut into pieces, and homogenized in 10 ml of lysis buffer [25 mM Tris/phosphate buffer, 8 mM MgCl2 (Wako), 1 mM DTT, 15% glycerol, and 1% TritonX-100 (Boehringer, Mannheim, Germany)]. The specimens were then rotated for 1 h at 4°C. After centrifugation, 10 µL of the supernatant was collected in a black 96-well flat-bottom plate (Corning, Corning, NY, USA), and 50 µL PicaGene Luminescence Kit (Toyo Ink, Tokyo, Japan) was added. Luciferase activity was assessed using a Spectramax M5, and the results were expressed in relative light units (RLU) per protein content (µg) in the supernatant. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
7
Trypsin-like Activity Assay Protocol
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Trypsin-like activity was assayed as described previously (Grenier, 1995 (link)). Trypsin-like activity was measured by monitoring the hydrolysis of the chromogenic synthetic peptide benzoyl-dl -arginine-p-nitroanilide (BAPNA; Peptide Institute) in the presence or absence of various compounds: EDTA (Wako Pure Chemical Industries), N-α-p-tosylamide-2-phenylethyl chloromethyl ketone (TLCK; Wako), leupeptin (Peptide Institute), DTT, iodoacetamide (Wako), SDS (Wako), CaCl2 (Sigma-Aldrich), MgCl2 (Wako) and ZnCl2 (Wako). The cells were harvested by centrifugation (10 000 g , 30 min) and suspended in distilled water. The bacterial samples (12.5 µl) were mixed with 125 µl of 150 mM Tris/HCl buffer (pH 7.8), 50 µl of 4 mM BAPNA and 12.5 µl distilled water, and the assay mixtures were incubated at 37 °C for 2 h. The release of p-nitroaniline was determined by measuring the OD405 nm using a microplate reader (Bio-Rad).
8
Cigarette Smoke Exposure Protocol
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The 3R4F cigarette was kindly provided by the Korea Institute of Toxicology. The TL and TW were purchased from Korean commercial sources. Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), 2-aminoanthracene, benzo[a]pyrene and cytochalasin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aroclor 1254-induced Sprague Dawley rat liver S9 was obtained from Moltox (Boone, NC, USA). The S9-cofactor, consisting of phosphate buffer, NADP, glucose 6-phosphate, KCl, MgCl2 and CaCl2, was purchased from Wako (Tokyo, Japan).
9
Detecting p38 MAPK Phosphorylation
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Phosphorylation of p38 MAPK was detected using Phos-tag gels containing 100 µM Phos-Tag acrylamide and 100 µM MgCl2 according to the manufacturer’s instructions (Wako Pure Chemical Industries).
10
Spinal Cord Protein Extraction and Analysis
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Mice were sacrificed, and the lumbar spinal cords were quickly dissected. The dissected tissue was homogenized in ice-cold buffer (20 mM Tris-HCl (Sigma, pH 7.4), 2% Triton X-100 (Sigma), 150 mM NaCl (FUJIFILM Wako), 1 mM EDTA (FUJIFILM Wako), 5 mM MgCl2 (FUJIFILM Wako), 10% anhydrous glycerol (FUJIFILM Wako), protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA)) and centrifuged. The extracts (20 µg of protein) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with a solution containing 3% skimmed milk and incubated overnight at 4 °C with anti-phospho-ERK1/2 (Thr202/Tyr204; 1:3000; #9101) (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2 (1:3000; #9102, Cell Signaling Technology), and anti-β-actin (1:3000; AC-74, Sigma) antibodies. The membranes were incubated with horseradish peroxidase-coupled anti-rabbit IgG sheep antibodies (GE Healthcare) for 1 h at room temperature. The reactive proteins were visualized using Luminata Forte HRP substrate (Merck Millipore, Nottingham, UK) according to the manufacturer’s instructions.
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