Fura 2 acetoxymethyl ester

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

Fura-2 acetoxymethyl ester is a fluorescent calcium indicator used for measuring intracellular calcium concentrations. It is a cell-permeant compound that can be easily loaded into cells.

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Lab products found in correlation

Metafluor software, by Molecular Devices (7 mentions) Pluronic f 127, by Thermo Fisher Scientific (4 mentions) Pluronic f 127, by Merck Group (4 mentions) Pluronic f 127, by Biotium (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Thrombin, by Enzyme Research (3 mentions) Ionomycin, by Merck Group (3 mentions) Fura 2 am, by Thermo Fisher Scientific (3 mentions) Nis element, by Nikon (3 mentions) Triton x 100, by Merck Group (2 mentions) Te2000 u, by Nikon (2 mentions) Te200, by Nikon (2 mentions) Gsk1016790a, by GlaxoSmithKline (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Dl dithiothreitol, by Merck Group (2 mentions) Indomethacin, by Merck Group (2 mentions) Igor pro, by Wavemetrics (2 mentions) Fluo 4 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions) Openlab software, by PerkinElmer (1 mentions) Igor pro software, by Wavemetrics (1 mentions)

82 protocols using fura 2 acetoxymethyl ester

Calcium Imaging of DRG Neurons

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DRG neurons were loaded with fura-2 acetoxymethyl ester (2 μM; Invitrogen, Carlsbad, CA) for 30 min at 37 °C in an atmosphere of 5% CO₂, as described previously24 (link). The ratio (R) of fluorescence signal measured at 340 nm, divided by the fluorescence signal measured at 380 nm, is proportional to [Ca²⁺]_i. Baseline [Ca²⁺]_i was determined from the average of five to eight measurements obtained before drug application. The agonists were applied alone, washed out, and then applied again in the continuous presence of NE. Amplitudes of peak [Ca²⁺]_i responses were computed as the difference between the peak value and the baseline value.

Zhu L., Zhao L., Qu R., Zhu H.Y., Wang Y., Jiang X, & Xu G.Y. (2015). Adrenergic stimulation sensitizes TRPV1 through upregulation of cystathionine β-synthetase in a rat model of visceral hypersensitivity. Scientific Reports, 5, 16109.

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Calcium Imaging in 2D and 3D hCS Cultures

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For calcium imaging in monolayer, hCSs were dissociated and cultured for two weeks. Cells were loaded with 1 μM Fura-2 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed with Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose and 25 mM HEPES, pH 7.4) and placed in a perfusion chamber on the stage of an inverted fluorescence microscope (TE2000U; Nikon). Imaging was performed at room temperature (23–25 °C) on an epifluorescence microscope equipped with an excitation filter wheel and an automated stage. The Openlab software (PerkinElmer) was used to collect and quantify time-lapse excitation ratio images, and fluorescence images were analyzed with the IGOR Pro software (WaveMetrics). For calcium imaging in 3D cultures, intact hCSs were loaded with 1 μM Fluo-4 acetoxymethyl ester (Invitrogen) for 30 min at 37 °C in Neurobasal supplemented with B-27, washed and sliced in half. Live imaging was performed at the NMS (Stanford Neuroscience Microscopy Service, supported by a US National Institute of Health grant, NS069375) using a Zeiss LSM780 confocal microscope.

Paşca A.M., Sloan S.A., Clarke L.E., Tian Y., Makinson C.D., Huber N., Kim C.H., Park J.Y., O’Rourke N.A., Nguyen K.D., Smith S.J., Huguenard J.R., Geschwind D.H., Barres B.A, & Paşca S.P. (2015). Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods, 12(7), 671-678.

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Calcium Imaging and Contractility Assays in Adult Cardiac Myocytes

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Ca²⁺ measurement assays in adult cardiac myocytes have been described previously [33 (link)]. In brief, cells were loaded with 2 µM Fura-2 acetoxymethyl ester (Invitrogen, C-2938) for 15 min, and then placed in Tyrode’s solution containing: 130 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose and 10 mM HEPES (pH 7.4). The Fura-2 fluorescence ratio was determined with a Delta scan dual-beam spectrofluorophotometer (Photon Technology International) operated at an emission wavelength of 510 nM and excitation wavelengths of 340 and 380 nM. The myocyte stimulation frequency for Ca²⁺ transient measurements was 0.5 Hz. For caffeine-induced Ca²⁺ release, myocytes were perfused with a Tyrode’s solution and stimulated at 0.5 Hz until stabilization of the transients, after which caffeine was acutely added. Ca²⁺ traces from healthy myocytes sensitive to caffeine treatment were processed using a Savitzky-Golay filter and baseline Ca²⁺ levels, transient amplitude, caffeine-induced Ca²⁺ release, and Ca²⁺ decay kinetics were analyzed using Felix 1.1 and Ion Wizard (IonOptix) software. For cell shortening measurements in isolated adult myocytes, cells were bathed in media 199 (M199) at room temperature using an Ionoptix system as described previously [34 (link), 35 (link)] and myocytes were electrically paced at 0.5 Hz with 60V and the solution was refreshed between each measurement.

Liu R., Correll R.N., Davis J., Vagnozzi R.J., York A.J., Sargent M.A., Nairn A.C, & Molkentin J.D. (2015). Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations. Journal of molecular and cellular cardiology, 87, 204-213.

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Chitin Modulates Platelet Ca2+ Signaling

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Ca²⁺ measurements were performed as described previously (Hussain and Mahaut-Smith, 1999 (link)). Briefly, 5 μM fura-2 acetoxymethyl ester (Invitrogen, France) and 0.2 μg/mL Pluronic F-127 were added to washed platelets pre-treated with different concentrations of chitin (20, 50, 100, or 200 μg/mL) in HBSS without Ca²⁺ at 37°C for 30 min. Platelets were then washed once and resuspended in HBSS. After platelet activation with thrombin (0.05 U/mL), the Ca²⁺ response was measured using a fluorometer (FLUOstar® Omega; BMG Labtech, Champigny sur Marne, France). The ratio of absorbance at 340/380 nm was converted into Ca²⁺ concentration (Ca²⁺).

Leroy J., Bortolus C., Lecointe K., Parny M., Charlet R., Sendid B, & Jawhara S. (2019). Fungal Chitin Reduces Platelet Activation Mediated via TLR8 Stimulation. Frontiers in Cellular and Infection Microbiology, 9, 383.

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Fura-2 Calcium Imaging in HEK-293 Cells

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For fluorescence imaging of [Ca²⁺]_i HEK-293 cells on poly-lysine-coated glass coverslips were loaded in standard bath solution containing membrane-permeable Fura-2 acetoxymethyl ester (1.5 ng/μl; Invitrogen) and pluronic acid (0,025%) for 20 min at 37°C. Fluorescence was alternately excited at 340 and 380 nm using the Polychrome IV monochromator (TILL Photonics). The emitted fluorescence was measured at 510 nm using a Sensicam (IMAGO). Fluorescence was corrected for background at each wavelength. Measurements were obtained at room temperature (21°C). The standard bath solution and stimulation with H₂O₂ were identical to those described for the patch-clamp experiments.

Kühn F.J., Kühn C, & Lückhoff A. (2015). Functional Characterisation of a TRPM2 orthologue from the sea anemone Nematostella vectensis in human cells. Scientific Reports, 5, 8032.

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Calcium Imaging of Astrocytes in CCI-ION

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For calcium imaging, primary astrocytes were loaded with Fura 2-acetoxy-methyl ester (10 μM, invitrogen) in darkness for 45 min at 37 °C. The cells were then transferred to a recording chamber (volume: 1.0 mL), continuously perfused with HEPES buffer at a flow rate of 1.5 ml/min at room temperature. CSF from either CCI-ION rats or sham rats was applied via bath application (100 μL). To ascertain astrocyte viability, HEPES containing 50 mM K⁺ was introduced at the end of each experiment. Fluorescence signals at 340 and 380 nm excitations were recorded every 2 s using an upright NIKON ECLIPSE Ti microscope equipped with a ratiometric imaging system (Nikon NIS-Elements AR 4.00.00, Japan). The ratio of fluorescence intensity at 340 nm and 380 nm [F_(340/380)] within a certain astrocyte was used to infer relative intracellular calcium concentration.

Yu N., Cui H., Jin S., Liu P., Fang Y., Sun F., Cao Y., Yuan B., Xie Y., Duan W, & Ma C. (2024). IL-6 from cerebrospinal fluid causes widespread pain via STAT3-mediated astrocytosis in chronic constriction injury of the infraorbital nerve. Journal of Neuroinflammation, 21, 60.

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Platelet activation signaling assays

Cited 2 times

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Thrombin was obtained from Enzyme Research Laboratories (South Bend IN, USA). Thrombin receptor-activating peptide 6 (SFLLRN, TRAP6) was purchased from Bachem (Bubendorf, Switzerland); cross-linked collagen-related peptide (CRP-XL) was from the University of Cambridge (Cambridge, UK); the stable ADP analogue methylthio-adenosine-diphosphate (Me-S-ADP) [8 (link)] came from Santa Cruz Biotechnology (Dallas TX, USA). Fura-2 acetoxymethyl ester and human fibrinogen were obtained from Invitrogen (Carlsbad CA, USA); Pluronic F-127 from Molecular Probes (Eugene OR, USA). Integrin α_IIbβ₃ inhibitor tirofiban [9 (link)] and cAMP-elevating agent iloprost [2 (link)] were from Sigma-Aldrich (St. Louis MI, USA); the selective Syk kinase inhibitor PRT-060318, 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide (Syk-IN) [10 (link)] came from Bio-Connect (Huissen, The Netherlands).

Zou J., Wu J., Roest M, & Heemskerk J.W. (2021). Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation. PLoS ONE, 16(3), e0247425.

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Fluorescence Imaging of [Ca2+]i in HEK-293 Cells

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For fluorescence imaging of [Ca²⁺]_i HEK-293 cells on poly-lysine-coated glass coverslips were loaded in standard bath solution containing membrane-permeable Fura-2 acetoxymethyl ester (1.5 ng/µl; Invitrogen) and pluronic acid (0,025%) for 20 min at 37 °C. Fluorescence was alternately excited at 340 and 380 nm using the Polychrome IV monochromator (TILL Photonics). The emitted fluorescence was measured at 510 nm using a Sensicam (IMAGO). Fluorescence was corrected for background at each wavelength. Measurements were obtained at room temperature (21 °C). The standard bath solution and the application of 2-APB were identical to those described for the patch-clamp experiments. In control experiments currents of hTRPM2 were evoked by superfusion of the cells with standard bath solution containing 10 mM H₂O₂ (diluted from a 30% stock solution).

Kühn F.J., Mathis W., Cornelia K., Hoffmann D.C, & Lückhoff A. (2017). Modulation of activation and inactivation by Ca2+ and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis. Scientific Reports, 7, 7245.

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Measuring Intracellular Calcium Dynamics

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[Ca²⁺]_c was measured using Fura-2. Cells were grown on glass coverslips and incubated 15 min at room temperature with 1 μM Fura-2 acetoxymethylester (Invitrogen Corp.Carlsbad, CA, USA,), 0.01% (v/v) Pluronic^® F-127 in standard extracellular solution (SES) (NIH 3T3 cells) or Krebs-Ringer-HEPES buffer (KRH2) (αT3 cells). SES contained (in mM): 140 NaCl, 4 KCl, 1 CaCl₂, 2 MgCl₂, 1 KH₂PO₄, 10 HEPES-NaOH (pH 7.4), and 10 d-glucose. KRH2 contained (in mM): 140 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 10 HEPES-NaOH (pH 7.4) and 2 d-glucose. Cells were placed into a temperature-controlled microperifusion chamber of an inverted fluorescence microscope (Olympus IX81, Olympus America, Inc., Center Valley, PA, USA). Cells were continuously superfused with SES or KRH2 (2 mL/min) at 37 °C. Fura-2 excitation wavelengths were 340 nm and 380 nm. The fluorescence emission intensities detected at 510 nm during excitation of Fura-2 at 340 nm (FI340) and 380 nm (FI380) were measured. MetaFluor software (Version 7.0, Molecular Devices LLC., San Jose, CA, USA) was used for image acquisition and analysis. Background fluorescence was subtracted from each FI340 nm and FI380 nm image. FI340/FI380 ratios were normalized to the average resting ratio 60 s prior to application of the stimulus.

Nelson H.A., Leech C.A., Kopp R.F, & Roe M.W. (2018). Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry. International Journal of Molecular Sciences, 19(5), 1522.

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Calcium Signaling Protocol in T Cells

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Cytosolic Ca²⁺ measurements were performed as previously described (15 (link)). Cells were serum starved in RPMI 1640 + L-glutamine supplemented with 0.5% BSA for 3 hours and plated on coverslips coated with poly-L-lysine or anti-CD3/CD28. Cells were loaded with 2 μM Fura-2 acetoxymethylester (Invitrogen) or 2 μM Fura-2NM acetoxymethylester (Teflabs) for 30 minutes at 25°C in cation-safe solution (107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl₂, 11.5 mM glucose, 20 mM Hepes-NaOH, 1 mM CaCl₂, pH 7.2). Cells were washed and dye allowed to de-esterify for 30 minutes at 25°C. Ca²⁺ measurements were made using a Leica DMI 6000B fluorescence microscope controlled by Slidebook Software (Intelligent Imaging Innovations; Denver, CO). Intracellular Ca²⁺ measurements are shown as 340/380 nm ratios obtained from groups of single cells. To measure PMCA-mediated Ca²⁺ clearance, cells were store depleted in 2 μM thapsigargin. Following store depletion, SOCE was induced by the addition of 1 mM Ca²⁺ and measured for 2 minutes, followed by Ca²⁺ clearance for 10 minutes in Ca²⁺-free imaging buffer + 100 nM EGTA.

Go C.K., Hooper R., Aronson M.R., Schultz B., Cangoz T., Nemani N., Zhang Y., Madesh M, & Soboloff J. (2019). Ca2+ export pump PMCA clears near-membrane Ca2+ to facilitate store-operated Ca2+ entry and NFAT activation. Science signaling, 12(602), eaaw2627.

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