Ca2 ionophore a23187

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany

Ca2+ ionophore A23187 is a chemical compound used as a research tool in laboratory experiments. It functions as a calcium (Ca2+) ionophore, which facilitates the transport of calcium ions across cellular membranes. The core function of Ca2+ ionophore A23187 is to enable the manipulation and study of calcium-mediated cellular processes in a controlled experimental setting.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Fluo 4 am, by Thermo Fisher Scientific (2 mentions) Accuri c6, by BD (2 mentions) Easysep mouse neutrophil enrichment kit, by STEMCELL (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Fetal calf serum (fcs), by Merck Group (2 mentions) Dmem media, by Merck Group (2 mentions) Ionomycin, by Alomone (1 mentions) Pluronic acid, by Thermo Fisher Scientific (1 mentions) Mibefradil, by Merck Group (1 mentions) Nnc 55 0396, by Merck Group (1 mentions) Bsa a7906, by Merck Group (1 mentions) Ethylene glycol bis 2 aminoethylether n n n n tetraacetic acid egta, by Merck Group (1 mentions) Nonidet p 40, by AppliChem (1 mentions) Bac to bac baculovirus expression system, by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by AppliChem (1 mentions) Indomethacin, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Ni nta agarose, by Qiagen (1 mentions) Waveguide, by GraphPad (1 mentions)

37 protocols using ca2 ionophore a23187

5-Lipoxygenase Metabolite Assay

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Freshly isolated
PMNL (5 × 10⁶) suspended in PBS pH 7.4 with 1 mg/mL
glucose or freshly withdrawn human whole blood were preincubated with
the test compounds for 10 min (or as indicated) at 37 °C. 5-LOX
product formation in PMNL was triggered by addition of arachidonic
acid (20 μM) and/or Ca²⁺-ionophore A23187 (2.5 μM;
Sigma-Aldrich) followed by incubation for 10 min at 37 °C. The
reaction was stopped with an equal volume of methanol.
Blood
was treated with Ca²⁺-ionophore A23187 (30 μM) for
10 min at 37 °C or was first primed for 30 min with LPS (1 μg/mL)
and then stimulated with fMLP (1 μM; Sigma-Aldrich) for 15 min.
After the reaction was stopped on ice, plasma was prepared (600g, 10 min, 4 °C), and aliquots were mixed with an equal
volume of methanol. Proteins were precipitated at −20 °C
for 2 h and removed by centrifugation (600g, 15 min,
4 °C).
Major 5-LOX metabolites (all-trans isomers of LTB₄ and
5-HETE) and, for PMNL, additionally 12-HETE (major 12-LOX product)
and 15-HETE (major 15-LOX product) were extracted and analyzed by
RP-HPLC as described for the determination of cell-free 5-LOX activity.
Zileuton (3 μM) was used as the reference 5-LOX inhibitor.

Neukirch K., Alsabil K., Dinh C.P., Bilancia R., Raasch M., Ville A., Cerqua I., Viault G., Bréard D., Pace S., Temml V., Brunner E., Jordan P.M., Marques M.C., Loeser K., Gollowitzer A., Permann S., Gerstmeier J., Lorkowski S., Stuppner H., Garscha U., Rodrigues T., Bernardes G.J., Schuster D., Séraphin D., Richomme P., Rossi A., Mosig A.S., Roviezzo F., Werz O., Helesbeux J.J, & Koeberle A. (2021). Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation. Journal of Medicinal Chemistry, 64(15), 11496-11526.

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Induced Egress of Toxoplasma Parasites

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Freshly egressed parasites were added to monolayers of HFFs in 12-well plates and allowed to grow for 20 h. Parasites were then cultured in media containing or lacking IAA for 16 h before 3 μM Ca²⁺ ionophore A23187 (Sigma-Aldrich, C9275-1MG-QM) was added to induce egress. After incubation in a water bath at 37°C for 5 min, parasites were fixed and an IFA was performed with rabbit anti-SAG2 and mouse anti-GRA7 antibodies. A total of 100 vacuoles were counted per field.

Fu J., Zhao L., Yang J., Chen H., Cao S, & Jia H. (2023). An unconventional SNARE complex mediates exocytosis at the plasma membrane and vesicular fusion at the apical annuli in Toxoplasma gondii. PLOS Pathogens, 19(3), e1011288.

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Calcium Signaling in Sperm Capacitation

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Chemicals were obtained from the following sources: bovine serum albumin (BSA) A7906, Ca²⁺ ionophore A23187, Mibefradil, NNC55-0396 and Ethylene glycol-bis (2-aminoethylether)-N,N,N,N′tetraacetic acid (EGTA) were purchased from Sigma–Aldrich Chemical Co. (St.Louis, MO); Fluo-4 AM and Fluo-3 AM from Molecular Probes, Thermo Fisher Scientific; Pluronic acid from Life Technologies Corporation (Invitrogen); PI from Santa Cruz (Santa Cruz, USA) and Ionomycin from Alomone Labs (Jerusalem, Israel). All other chemicals were of reagent grade. Fluo-4 AM, Fluo-3 AM, Pluronic acid, Ca²⁺ ionophore A23187 and Ionomycin were dissolved in DMSO; EGTA was dissolved in non-capacitating modified TYH medium without Ca²⁺ (-HCO₃⁻, -BSA, -Ca²⁺); while PI, Mibefradil and NNC55-0396 were dissolved in hexa-distilled water.

Luque G.M., Dalotto-Moreno T., Martín-Hidalgo D., Ritagliati C., Puga Molina L.C., Romarowski A., Balestrini P.A., Schiavi-Ehrenhaus L.J., Gilio N., Krapf D., Visconti P.E, & Buffone M.G. (2018). Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation. Journal of cellular physiology, 233(12), 9685-9700.

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Enzyme Activity Characterization Protocol

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GA (purity ≥ 98%) was a gift from Dr. Willmar Schwabe GmbH & Co. KG (Karslruhe, Germany). Zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea] was from Sequoia Research Products (Oxford, UK); PGH₂ was from Larodan (Malmö, Sweden); IL-1β was from ReproTech (Hamburg, Germany); RSC-3388 was from Calbiochem (Darmstadt, Germany); EDTA and Nonidet P-40 were from AppliChem (Darmstadt, Germany); p-anisidinium chloride was from Merck (Darmstadt, Germany); Insect Express Sf9-S2 and RPMI media, glutamine, penicillin, and streptomycin were from PAA (Coelbe, Germany); Bac-to-Bac baculovirus expression system was from Invitrogen (Karlsruhe, Germany); Ni-NTA agarose was from Qiagen (Hilden, Germany). AA, Ca²⁺-ionophore A23187, dextrane, dithiothreitol, fetal calf serum, indomethacin, lipopolysaccharide (LPS), Triton X-100, and all other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. HPLC/UPLC solvents were from VWR (Darmstadt, Germany).

Gerstmeier J., Seegers J., Witt F., Waltenberger B., Temml V., Rollinger J.M., Stuppner H., Koeberle A., Schuster D, & Werz O. (2019). Ginkgolic Acid is a Multi-Target Inhibitor of Key Enzymes in Pro-Inflammatory Lipid Mediator Biosynthesis. Frontiers in Pharmacology, 10, 797.

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OVA-specific IgE Calcium Signaling

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5 × 10⁵ LAD2 cells were sensitized overnight with PBS or 500 ng/mL OVA-specific ^SiahIgE or ^AshIgE. Next day, sensitized cells were washed before loading with 2 μM Fluo-4-AM (Invitrogen) at 37°C in HEPES buffer for 20 minutes. After loading, the cells were washed and resuspended in HEPES buffer. Fluorescence was filtered through the 530/30 band pass filter and collected in FL-1/FITC. Baseline Ca²⁺ fluorescence levels were recorded for 1 minute on the Accuri C6 (BD Biosciences) before the addition of indicated allergen or buffer to each sample. At the end of allergen stimulation, cells were added 2 μM Ca²⁺ ionophore A23187 (Sigma) as a positive control.

Shade K.T., Conroy M.E., Washburn N., Kitaoka M., Huynh D.J., Laprise E., Patil S., Shreffler W, & Anthony R.M. (2020). IgE Sialylation is a Determinant of Allergic Pathogenicity. Nature, 582(7811), 265-270.

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Ca2+ Mobilization Assay for Agonists

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During the 1^st-addition of the Ca²⁺-mobilization assay described above, cells were exposed to either vehicle-only (DMSO; 0.1% final concentration) or 13-point three-fold dilutions of OT, Ca²⁺ ionophore A23187 (Sigma) and PGF_2α (starting at 100uM final concentration). For each well, the mean baseline value (MBV) for Ca²⁺-fluorescence was calculated during the 0–19 sec timeframe. The max RFU was calculated from the 20–140 sec timeframe, and the baseline was subtracted from the max RFU. An average Max-MBV RFU was calculated for each concentration of agonist (OT, A23187 and PGF_2α). Data were analyzed using WaveGuide and GraphPad Prism 6. Non-linear regression analyses were performed to generate concentration response curves and to determine the E_max and EC₅₀ value for each experimental day.

Siricilla S., Knapp K.M., Rogers J.H., Berger C., Shelton E.L., Mi D., Vinson P., Condon J., Paria B.C., Reese J., Sheng Q, & Herington J.L. (2019). Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery.. Pharmacological research, 146, 104268.

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Neutrophil Stimulation and MPO Assay

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Mouse neutrophils were isolated from the spleen using EasySep, mouse Neutrophil Enrichment Kit (Cat# 19762; Stem cell technologies, Cambridge, MA). Cells were seeded in a 48-well plate (200000 cells/well) in 100 μL of HBSS (10 mM HEPES with calcium and magnesium) with 0.2% rat serum and incubated for 15 min at 37°C. Cells were stimulated by adding an equal volume of 4 μM Ca²⁺ ionophore A23187 (#C7522 Sigma Aldrich). After 4 h incubation, 0.5 U of micrococcal nuclease was added to the wells, swirled, and incubated for 10 min at 37°C. Supernatants were collected and adjusted to 2 mM ethylenediaminetetraacetic acid (EDTA), clarified by centrifugation at 5,000 x g for 5 min, and used for MPO assay. MPO activity was measured by oxidation of tetramethylbenzidine (TMB, 2 mM) in the presence of H₂O₂, followed by spectrophotometry.

Meena A.S., Shukla P.K., Bell B., Giorgianni F., Caires R., Fernández-Peña C., Beranova S., Aihara E., Montrose M.H., Chaib M., Makowski L., Neeli I., Radic M.Z., Vásquez V., Jaggar J.H., Cordero-Morales J.F, & Rao R. (2022). TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response. Cell reports, 39(11), 110937.

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HeLa Cell Culture and Treatment

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HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) in 5% CO₂ in a humidified environment at 37°C as previously described [7] (link), [15] (link). In most assays, the 100-mm culture dishes were seeded with 1×10⁶ cells in 5 ml of growth medium and allowed them to expand to about 85% to 90% confluence. Cell treatments were performed with the following compounds: Ca²⁺ ionophore A23187 (Sigma), paclitaxel and cisplatin (BioVision), and inhibitor ALLN or MG132 (BioVision).

Fan X., Zhang Q., You C., Qian Y., Gao J., Liu P., Chen H., Song H., Chen Y., Chen K, & Zhou Y. (2014). Proteolysis of the Human DNA Polymerase Delta Smallest Subunit p12 by μ-Calpain in Calcium-Triggered Apoptotic HeLa Cells. PLoS ONE, 9(4), e93642.

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Measuring SERCA ATPase Activity in Muscle

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To examine SERCA function in response to BSO treatment, aging, and fatiguing stimulation, Ca²⁺-dependent ATPase activity was measured using a spectrophotometric plate reader assay at 37°C as described previously (Duhamel et al. 2007 (link)). Briefly, muscle homogenates were diluted in reaction buffer containing in mmol/L: 200 KCl, 20 HEPES, pH 7.0, 10 NaN₃, 1 EGTA, 15 MgCl₂, 10 phosphoenolpyruvate, 5 ATP, 1 Ca²⁺-ionophore A-23187 (Sigma), as well as 18 U/mL of both lactate dehydrogenase and pyruvate kinase. The mixture was divided in five equal aliquots with pCa ranging from 7.0 to 5.0. Samples were loaded in duplicate into a 96-well plate and the reaction was initiated by the addition of 0.3 mmol/L NADH to each well. ATPase activity was also assessed in the presence of 130 μmol/L cyclopiazonic acid, a highly specific SERCA inhibitor (Goeger et al. 1988 (link); Seidler et al. 1989 (link); Inesi and Sagara 1994 (link)), to determine background ATPase activity. Maximal SERCA activity was determined for each sample as the difference between the total ATPase activity of the group of wells with the highest ATPase activity and the background ATPase activity measured with cyclopiazonic acid, and then normalized to total protein concentration.

Smith I.C., Vigna C., Levy A.S., Denniss S.G., Rush J.W, & Tupling A.R. (2015). The effects of buthionine sulfoximine treatment on diaphragm contractility and SERCA pump function in adult and middle aged rats. Physiological Reports, 3(9), e12547.

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Age-Dependent SERCA ATPase Activity Assay

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The measurement of SERCA ATPase activity was performed at 37°C using a spectrophotometric assay as described elsewhere.27 The gastrocnemius muscle, which has been shown to be particularly responsive to age and oxidative stress‐related changes in mass and force generation, was used for this analysis. Briefly, muscle samples (15–20 mg) were diluted 1:10 (wt/vol) and manually homogenized in ice‐cold homogenizing buffer (pH 7.5) containing (in mM) 250 sucrose, 5 HEPES, 0.2 PMSF, and 0.2% sodium azide (NaN₃). Ca²⁺‐dependent Ca²⁺‐ATPase activity was measured in the assay buffer containing 100 mM KCl, 20 mM HEPES, pH 7.0, 10 mM MgCl₂, 10 mM NaN3, 10 mM phosphoenolpyruvate, 1 mM EGTA, 5 mM ATP, 18 U/mL of both lactate dehydrogenase and pyruvate kinase, and 1 mM Ca²⁺ ionophore A‐23187 (C‐7522, Sigma). The reaction mixture was divided into 10 aliquots of 300 μL and mixed with CaCl₂ to generate 10 different Ca²⁺concentrations, ranging between 7.6 and 5 pCa units. The reaction was started by adding 0.3 mM NADH. Basal activity was determined in the presence of 40 μM of the Ca²⁺‐ATPase inhibitor cyclopiazonic acid (C‐1530, Sigma) in dimethyl sulfoxide.

Qaisar R., Bhaskaran S., Premkumar P., Ranjit R., Natarajan K.S., Ahn B., Riddle K., Claflin D.R., Richardson A., Brooks S.V, & Van Remmen H. (2018). Oxidative stress‐induced dysregulation of excitation–contraction coupling contributes to muscle weakness. Journal of Cachexia, Sarcopenia and Muscle, 9(5), 1003-1017.

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