Fetal bovine serum (FBS) was obtained from HyClone; GE Healthcare Life Sciences (Logan, UT, USA), and all other cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Spermine (a CaSR agonist), Calhex231 (a CaSR negative allosteric modulator), MRS1845 (a SOCC inhibitor) and SKF96365 (a TRPC inhibitor) were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Rabbit anti-TRPC1 monoclonal antibody (catalog no. ACC-010) was obtained from Alomone Laboratories, Ltd. (Jerusalem, Israel). Polyclonal mouse anti-human CaSR antibody was obtained from Abcam (Cambridge, MA, USA; catalog no. ab62653, for western blotting) and from Shanghai Seebio Science & Technology Co., Ltd. (Shanghai, China; catalog no. HL1499 for immunohistochemistry) and other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Small interfering RNA (siRNA) was purchased from Yangzhou Ruibo Biotech Co., Ltd. (Yangzhou, China). Lipofectamine™ 2000, Fura-2-acetoxymethyl ester (AM) and the NO Fluorescence kit were obtained from Invitrogen; Thermo Fisher Scientific, Inc.
Fura 2 acetoxymethyl ester am
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fura-2 acetoxymethyl ester (AM) is a fluorescent calcium indicator used for the quantitative measurement of intracellular calcium concentrations. It is a cell-permeable derivative of the Fura-2 dye that can be loaded into cells and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Spermine, by Merck Group (1 mentions)
Other antibodies, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Calhex231, by Merck Group (1 mentions)
Skf96365, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
L phe, by Merck Group (1 mentions)
Nps 2143, by Bio-Techne (1 mentions)
Skf96365, by Bio-Techne (1 mentions)
Pyr10, by Merck Group (1 mentions)
2 apb, by Bio-Techne (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Pdgf aa, by R&D Systems (1 mentions)
Human recombinant fgf2, by R&D Systems (1 mentions)
Recombinant human insulin, by Fujifilm (1 mentions)
Naltriben, by Merck Group (1 mentions)
3 isobuthyl 1 methylxanthine, by Merck Group (1 mentions)
Ns8593, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
16 protocols using fura 2 acetoxymethyl ester am
1
Regulation of Calcium Signaling in Cells
2
Biochemical Reagents for Cell Signaling
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Acz, L-Phe, Pyr6 and Pyr10 were purchased from Sigma-Aldrich (St. Louis, MO, USA) whereas Pyr3, 2-APB, NPS-2143, and SKF-96365 were purchased from Tocris Bioscience (Minneapolis, MN, USA). All cell culture media including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), glutamine, and Fura-2/acetoxymethyl ester (AM) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals used in this study were analytical grade.
3
Fluorescence Imaging of Calcium Dynamics in Islet Cells
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Mouse islet clusters were prepared and transfected with OPN shRNA or an empty control plasmid as outlined in the previous section. The cells were loaded with 2 μM Fura-2-acetoxymethyl ester (AM) (Thermo Fisher Scientific) for 25 min at 37°C, 5% CO2, washed twice with KRHB with 2.0 mM glucose, and incubated 20 min in KRHB with 2.0 mM glucose at 37°C, 5% CO2. Fluorescence imaging was performed using a Nikon Eclipse TE2000-U microscope and the data was analyzed using Nikon Elements software. Cells were perifused at 37°C with a flow of 2 mL/min KRHB with 2.0 mM glucose for 2 min then perifused under identical conditions with KRHB with 11.0 mM glucose for 20 min. The ratios of emitted fluorescence intensities at excitation wavelengths of 340 and 380 nm (F340/F380) were recorded every 5 seconds.
4
Myelination Regulation in Oligodendrocytes
5
Intracellular Calcium Signaling Assay
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2‐aminoethyl diphenylborinate (2‐APB), N‐methyl maleimide (NMM), NS8593, naltriben, dexamethasone (DEX), and 3‐isobuthyl‐1‐methylxanthine (IBMX) were purchased from Sigma‐Aldrich (St. Louis, MO). 2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐1,3‐propanediol (FTY720) was purchased from Cayman Chemicals (Ann Arbor, MI). Recombinant human insulin was purchased from Wako (Osaka, Japan). Fura‐2 acetoxymethyl ester (AM) was purchased from Thermo Fisher Scientific (Waltham, MA).
6
Intracellular Calcium Imaging in HUVEC Cells
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Intracellular Ca2+ was determined using fluorescent Ca2+ indicator fura-2-acetoxymethyl ester (AM) (Molecular probes, Seoul, Korea). HUVEC cells were seeded at 5 × 104/ well in a 0.1% gelatin-coated black 96-well plate (Corning, NY, USA) overnight. Then, cells were washed with Krebs-Ringer HEPES (KRH) buffer, including 138 mM NaCl2, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgCl2, 24 mM NaHCO3, 2 mM KH2PO4, 10 mM Na2HPO4, 25 mM HEPES, and 10 mM glucose. After washing with KRH buffer, cells were incubated with fura-2 AM (4 µM) for 30 min in KRH buffer without CaCl2, then washed, left for 30 min to allow complete de-esterification of fura-2 AM in complete KRH solution. For drug effects, zileuton (50 µM) or NS1619 (10 µM) in the presence or absence of VEGF (10 ng/mL) were added then, intracellular Ca2+ influx was measured. Intracellular Ca2+ rise was monitored by measuring the ratio of 510 nm emission using the Nikon TS 100 fluorescence imaging system (Nikon Instrument Inc., USA) according to the manufacturer’s guideline and changes in intracellular Ca2+ fluctuation as function of time were plotted and analyzed by the InCyt Im2 software (University of Cincinnati, USA).
7
Cell Culture and Calcium Imaging
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Dulbecco’s modified eagle media (DMEM), fetal bovine serum (FBS), and horse serum (HS) were obtained from Gibco-BRL (Grand island, NY, USA). Fura-2 acetoxymethyl ester (AM) was purchased from Molecular Probes (Eugene, OR, USA). 6-Methyl-2-(phenylethynyl) pyridine hydrochloride, 3,5-dihydroxyphenylglycine (DHPG), and other chemicals were purchased from Sigma (St. Louis, MO, USA).
8
Measuring Bacterial Calcium Levels
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Calcium levels were measured with fura-2 acetoxymethyl ester (AM) (Molecular Probes) following the methodology reported by Refs.29 (link),30 (link) with some modifications. Bacterial cells 1 × 107 CFU/mL were incubated with 5 ppm of ARGIRIUM-SUNCs or 30% v/v of H2O2 (control samples) for 2 h at 37 °C. After that, cells were washed twice in Krebs Buffer 7 Fura-2AM fluorescence was recorded at 340/380 nm excitation and 512 nm emission. The peak amplitude of Fura-2AM fluorescence (ratio at 340/380 nm) was used to evaluate ER calcium levels.
9
Calcium Signaling Pathway Modulation
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Materials were purchased from the following companies: fura-2 acetoxymethyl ester (AM) from Molecular Probes (Eugene, OR, USA); Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA, USA); the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide (NBQX), the L-type Ca2+ channel antagonist nimodipine, PKCε translocation inhibitor and PKCζ pseudosubstrate inhibitor, CaMKII inhibitor KN-62 from Calbiochem (San Diego, CA, USA); the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5), the intracellular calcium chelator BAPTA, 1,2-bis(2-aminophenoxy)ethane-*N,N,N',N'-tetraacetic acid; the nonspecific PKC inhibitor staurosporin, the JAK-2 inhibitor AG490, the MAPK kinase inhibitor PD98059 and all other reagents from Sigma (St. Louis, MO, USA). Mg2+ free-DMEM (DMEM without MgSO4) was purchased from WelGENE (Korea) as a customer-ordered item.
10
Intracellular Calcium Measurement in VSC4.1 Neurons
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The intracellular [Ca2+] in VSC4.1 neurons was measured with the Ca2+-sensitive indicator Fura-2/acetoxymethyl ester (AM) (Molecular Probes, CA, USA) [14 (link)]. After each treatment, the neurons were loaded with 5 μM Fura-2-AM for 30 min at 37°C in the dark. After dilution to 1 × 106 cells/mL with the same Ca2+ buffer solution, Fura-2-AM was excited at wavelengths of 340 and 380 nm. The relative changes in intracellular [Ca2+] were determined by the fluorescence ratio (R) at 340/380 with the following formula: [Ca2+] = Kd × β × (R − Rmin)/(Rmax − R) [14 (link)]. The Calcium Calibration Buffer Kit with Magnesium (Molecular Probes, CA, USA) was used to determine that the Kd, a cell-specific constant, for VSC4.1 neurons was 0.264 μM.
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