Egta am

Manufactured by Thermo Fisher Scientific

Sourced in United States

EGTA-AM is a calcium chelating agent that can be used to study calcium signaling in biological systems. It is a cell-permeable derivative of EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), a widely used calcium chelator. EGTA-AM can permeate cell membranes and then be hydrolyzed by intracellular esterases to release the active EGTA chelator, which can bind and sequester calcium ions.

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Lab products found in correlation

Bapta am, by Thermo Fisher Scientific (2 mentions) Pluronic f 127, by Thermo Fisher Scientific (2 mentions) Ml si1, by Enzo Life Sciences (1 mentions) Recombinant mouse m csf carrier free, by BioLegend (1 mentions) Paxilline, by Cayman Chemical (1 mentions) Ml sa1, by Bio-Techne (1 mentions) Ns1619, by Merck Group (1 mentions) Bapta am, by Bio-Techne (1 mentions) 4 methylumbelliferyl n acetyl β d glucosaminide, by Merck Group (1 mentions) Gsk1016790a, by Merck Group (1 mentions) Probenecid, by Merck Group (1 mentions) 8 cyclopentyltheophylline 8 cpt, by Merck Group (1 mentions) Cal 520, by AAT Bioquest (1 mentions) Fluo 8 am, by AAT Bioquest (1 mentions) Pclamp 9, by Molecular Devices (1 mentions) Axioskop 2, by Zeiss (1 mentions) Multiclamp 700b, by Molecular Devices (1 mentions) Gabazine sr 95531, by R&D Systems (1 mentions) Resiniferatoxin rtx, by R&D Systems (1 mentions) Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

NP-EGTA AM (2 citations)

13 protocols using egta am

Immunofluorescence Staining of Slo1 and Lamp1

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The following primary antibodies were used for immunofluorescence staining: anti-Slo1 (UC Davis/NIH NeuroMab Facility, Davis, CA or Abcam, Cambridge, UK), anti-Lamp1 (H4A3, 1D4B, Developmental Studies Hybridoma Bank, USA), Alexa 488 goat anti-rat antibody, Alexa 488 goat anti-rabbit antibody, Alexa 546 goat anti-mouse antibody and Texas Red goat anti-mouse antibodies were purchased from Invitrogen (Invitrogen, USA). The following chemicals were also used: Paxilline (Cayman Chemical Company, USA); TEA (Sigma, USA); ML-SA1 (Tocris Bioscience, UK); ML-SI1 (Enzo Life Sciences Inc, USA); NS1619 (Sigma, USA); BAPTA-AM (Tocris Bioscience, UK); EGTA-AM (Invitrogen, USA); Recombinant Mouse M-CSF (carrier-free) (BioLegend, USA); 4-Methylumbelliferyl N-acetyl-β-d-glucosaminide (Sigma, USA).

Sun X., Xu M., Cao Q., Huang P., Zhu X, & Dong X.P. (2020). A lysosomal K+ channel regulates large particle phagocytosis by facilitating lysosome Ca2+ release. Scientific Reports, 10, 1038.

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Hypotonic Challenge and TRPV4 Modulation

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Hypotonic challenges were achieved by dilution of media with dH₂O to 85 mOsm/kg H₂O; osmolality was measured using an Osmomat 3000 freezing point depression osmometer (Gonotec). Inhibitors were used at 100× published IC₅₀ values and were added 30 min prior to initiation and maintained for the duration of each experimental setting. TRPV4 inhibitor (HC‐067047) was used at a concentration of 4.8 μm, while calmodulin antagonist (trifluoperazine, TFP) was used at a concentration of 127 μm. The potent TRPV4 channel agonist GSK1016790A (Sigma) was used at a concentration of 2.1 μm; it was added either 30 min before the intervention or at the same time as the intervention. Intracellular calcium chelation was performed using EGTA AM (Invitrogen; E1219) at a concentration of 5 μm following the manufacturer's recommendation. In all cases, cells were at least 92% viable, assessed using CellTiter‐Glo^® Luminescent Cell Viability Assay.

Salman M.M., Kitchen P., Woodroofe M.N., Brown J.E., Bill R.M., Conner A.C, & Conner M.T. (2017). Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)‐ and calmodulin‐mediated mechanism. The European Journal of Neuroscience, 46(9), 2542-2547.

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Preparation of Pharmacological Agents for Electrophysiology

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Drug solutions were prepared fresh at the start of each experiment to prevent degradation by environmental factors e.g. light. Solutions were made using de-ionized water (pH 5–6, resistance 18.2 MΩcm) from a Milli-Q UV plus system.
ChTX was dissolved in water and stored in small aliquots at −20°C. It was then dissolved to its final concentration of 10 nM. The sodium channel blocker, tetrodotoxin (TTX, 1 μM), was used to block initiation and propagation of action potentials. The selective A1 –receptor antagonist, 8-cyclopentyltheophylline (8-CPT; Sigma, St. Louis, MO), was initially dissolved in DMSO and subsequently in ACSF to give a final concentration of 10 μM.
Bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EGTA-AM (Molecular Probes, Eugene, OR) were initially dissolved in DMSO, and then diluted to their final concentrations in the ACSF. DMSO concentration in ACSF was 0.0001% for 1 μM BAPTA-AM and 0.00006% for 50 μM of EGTA-AM. The chelator freely entered the cell due to the AM moiety and was then deesterified to cell-impermeant BAPTA. Probenecid (Sigma, St. Louis, MO) was dissolved in 1 M NaOH and subsequently buffered with HCl acid to pH 7.4. Whenever Probenecid was used (1 mM) care was taken to adjust the sodium concentration of the ACSF. Probenecid and DMSO did alone did not alter fEPSP at concentrations used.

Jalini S., Ye H., Tonkikh A.A., Charlton M.P, & Carlen P.L. (2016). Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission. PLoS ONE, 11(3), e0148110.

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Intracellular Calcium Imaging Reagents

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The membrane-permeant caged IP₃ analog ci-IP₃/PM (D-2,3-O-Isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-Inositol 1,4,5-trisphosphate-Hexakis (propionoxymethyl) Ester) was obtained from SiChem (Bremen, Germany), diluted in 20% pluronic F-127 solution in dimethylsulfoxide to a stock concentration of 200 μM and was frozen down into 2-μl aliquots until needed. EGTA-AM and pluronic F-127 were from Molecular Probes/Invitrogen (Carlsbad, CA, USA). Fluo-8 AM and Cal520 were purchased from AAT Bioquest (Sunnyvale, CA, USA).

Schmunk G., Boubion B.J., Smith I.F., Parker I, & Gargus J.J. (2015). Shared functional defect in IP3R-mediated calcium signaling in diverse monogenic autism syndromes. Translational Psychiatry, 5(9), e643-.

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Electrophysiological recordings of NTS neurons

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Patch electrodes (2–3.5 MΩ) were pulled from borosilicate glass and filled with an intracellular solution composed of (mM): 6 NaCl, 4 NaOH,130 K-Gluconate, 11 EGTA, 2 CaCl₂, 2 MgCl₂, 10 HEPES; 2 Na₂ ATP, 0.2 Na₂ GTP; pH adjusted to 7.3 – 7.32. Neurons were visualized using infrared differential interference contrast optics (Zeiss Axioskop2) and selected for recording within the medial NTS (250 μm rostral of obex and medial to the ST). Neurons were voltage clamped to −60 mV (Multiclamp 700B) and currents were sampled at 20 kHz and digitally filtered at 10 kHz with pClamp 9.2 software (Axon Instruments, Union City, CA). Gabazine (SR-95531, 3 μM, R&D Systems) was added to all ACSF to block GABA_A synaptic currents. To classify ST inputs, the potent TRPV1 selective agonist, resiniferatoxin (RTX, 2nM, R&D Systems) was superfused at the end of each experiment (Doyle et al., 2002 (link)). All other drugs were applied as indicated: 5-HT₃R specific agonist, m-chlorophenylbiguanide HCl (PBG, 1 or 10 μM, Sigma Aldrich); 5-HT₃R specific antagonist Ondansetron HCl (OND, 0.5 μM R&D Systems); cell-permeable calcium chelator EGTA-AM (10 μM, Molecular Probes).

Fawley J.A., Doyle M.W, & Andresen M.C. (2019). 5-HT3R–sourced Calcium Enhances Glutamate Release from a Distinct Vesicle Pool. Brain research, 1721, 146346.

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Protein Trafficking and Calcium Signaling

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Thapsigargin, filipin, CaCl₂, EDTA, and cholesterol were purchased from Sigma. EGTA-AM was purchased from Thermo Scientific. Dynasore was purchased from Abcam. Miglustat was purchased from Tocris. Antibodies used in the study were: mouse anti-α-tubulin (Abcam); rat anti-LAMP1 (Clone 1D4B, Development Studies Hybridoma Bank), mouse anti clathrin heavy chain (BD Biosciences), mouse anti-STIM1 (Cell Signalling), rabbit anti-TFEB (Proteintech), rabbit anti-calcineurin (Abcam), rabbit anti-histone H3 (Cell Signalling), and rabbit anti SREBP (Abcam). For immunoblotting, the secondary antibodies were conjugated to horseradish peroxidase (Jackson Laboratories) or fluorochromes (IR-dye 800 or IR-dye 680; LI-COR). Secondary antibodies used for immunofluorescence were purchased from Thermo Scientific.

Boutry M., Pierga A., Matusiak R., Branchu J., Houllegatte M., Ibrahim Y., Balse E., El Hachimi K.H., Brice A., Stevanin G, & Darios F. (2019). Loss of spatacsin impairs cholesterol trafficking and calcium homeostasis. Communications Biology, 2, 380.

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Comprehensive Calcium Signaling Assay

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Apamin, cyclopiazonic acid (CPA), GSK2193874, GSK1016790A, HC067047, NS309, 1400W, Nω‐propyl‐l‐arginine hydrochloride, ODQ, RN1747, Rp‐8‐Br‐PET‐cGMPS, Suramin, and Tram 34 were purchased from Tocris Bioscience (Minneapolis, MN). ATP and N^ω‐nitro‐l‐arginine were obtained from Sigma‐Aldrich (St. Louis, MO). DAF‐FM diacetate, Fluo‐4AM (Ca²⁺ indicator), and EGTA‐AM (Ca²⁺ chelator) were purchased from Fischer Scientific (Pittsburgh, PA). Spermine NONOate and U46619 were purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were obtained from Sigma‐Aldrich (St. Louis, MO).

Marziano C., Hong K., Cope E.L., Kotlikoff M.I., Isakson B.E, & Sonkusare S.K. (2017). Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(12), e007157.

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Intracellular Ca2+ and IP3 Signaling Assay

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Cells were incubated with 5 μM of the membrane-permeant fluorescent Ca²⁺ indicator Cal520-AM (AAT Bioquest #21130; Sunnyvale, CA) for 1 hr at room temperature in a Ca²⁺-containing HEPES buffered salt solution (Ca²⁺-HBSS) together with 5 μM of the membrane-permeant ester of the caged IP₃ analogue ci-IP₃-PM (D-2,3,-O-Isopropylidene-6-O-(2-nitro-4,5 dimethoxy) benzyl-myo-Inositol 1,4,5,-trisphosphate hexakis (propionoxymethyl) ester) (SiChem #cag-iso-2–145-10; Bremen, Germany). In some experiments cells were additionally loaded by incubation with EGTA-AM (15 μM; Thermo Fisher Scientific #E1219) for a further 1 hr at room temperature in Ca²⁺-HBSS. Following loading, cells were maintained for 30 min at room temperature in Ca²⁺-HBSS before imaging. Cal520-AM, ci-IP₃-PM, and EGTA-AM were all solubilized with DMSO/20% pluronic F127 (ThermoFisher #P3000MP). Ca²⁺-HBSS contained (in mM) 135 NaCl, 5.4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH=7.4). Zero Ca²⁺-HBSS consisted of the same formulation with CaCl₂ omitted, and 300 μM EGTA added. The receptor agonists carbachol (#C4832) and histamine (#H7125) were purchased from Millipore Sigma and solubilized in zero Ca²⁺-HBSS.

Vorontsova I., Lock J.T, & Parker I. (2022). KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters. Cell calcium, 107, 102638.

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Inducing CREB Phosphorylation in Cortical Neurons

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To induce CREB phosphorylation, we stimulated cortical neurons with the indicated high [K⁺] solution at 37 °C for 10–300 s, and fixed the cells immediately after the stimulation (in 4% paraformaldehyde in PBS, with 20 mM EGTA and 4% (w/v) sucrose), or incubated the cells in culture media for 90 min at 37 °C before fixation. Where indicated, drugs were added 30 min before and included throughout the stimulation. All K⁺-rich stimulation solutions contained 0.5 μM TTX (Ascent Scientific) to block action potentials. In addition, when stimulating cortical neurons, 10 μM NBQX (Ascent Scientific) and 10 μM APV (Ascent Scientific) were included to block AMPA and NMDA receptors, respectively. 4 mM K Tyrode’s consisted of (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, pH 7.4. When stimulating with elevated [K⁺], Na⁺ was adjusted to maintain osmolarity. To block: CaM Kinases, KN93 (Tocris) was used at 4uM; to block Ca_V1 channels, Nimodipine (abcam) was used at 10uM; to block CaMKK, STO609 (Tocris) was used at 3.3uM; to block PP2A, okadaic acid (Tocris) was used at 20 nM; to block PP1 and PP2A, okadaic acid (Tocris) was used at 2uM.
Cells were loaded with Ca²⁺ chelators EGTA-AM and BAPTA-AM (Life Technologies), used at 200uM, along with 1:1000 Pluronic F-127 (Life Technologies) 45 min before stimulation.

Cohen S.M., Li B., Tsien R.W, & Ma H. (2015). Evolutionary and functional perspectives on signaling from neuronal surface to nucleus. Biochemical and biophysical research communications, 460(1), 88-99.

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Blocking Ca2+ Signaling Pathways

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Unless otherwise noted, drugs were dissolved in amphibian saline solution and superfused over the slice preparations. Cadmium (CdCl₂, 100–500 μM Sigma Chemicals) was used to block influx through Ca²⁺ channels. The ryanodine receptor blocker, dantrolene (10 μM), was used to block Ca²⁺ release from internal stores (Chen et al., 2014 (link)). Due to concerns about incomplete recovery after washout, each slice preparation was exposed to Cd²⁺ only once and then new slices were prepared for subsequent experiments. In some experiments, retinal slices were incubated with 100 μM EGTA-AM (Life Technologies) for 2 hours to increase intracellular Ca²⁺ buffering prior to starting electrophysiological recordings

Cork K.M., Van Hook M.J, & Thoreson W.B. (2016). Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors. The European journal of neuroscience, 44(3), 2015-2027.

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