2 apb

Manufactured by Cayman Chemical

Sourced in United States

2-APB is a chemical compound commonly used in research laboratories. It functions as a selective 5-HT2B receptor agonist, which means it has the ability to activate the 5-HT2B receptor subtype. The core function of 2-APB is to serve as a research tool for studying the 5-HT2B receptor and its related biological processes.

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8 protocols using 2 apb

Fractalkine Modulation of Microglia

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BV-2 cells (Cell Resource Centre of the Beijing Union, China), a murine microglial cell line, were cultured in DMEM/F12 containing 10% fetal bovine serum and 1% penicillin and streptomycin, at 37°C with 5% carbon dioxide. When cultures were 70–80% confluent, and 0.25% trypsin was used to digest and passage cells. Cells were seeded in 35-mm-diameter dishes (5 mL/dish), 24-well plates (1 mL/well), or 6-well plates (2 mL/well) at a density of 1×10⁵ cells/ml. After 24 h of growth, culture medium was replaced with serum-free DMEM/F12 medium and synchronized for 12 h.
Cells were randomly divided into 4 groups (n=18 per group): control (C), fractalkine (aa25-105, R&D Systems, Minneapolis, MN) (F), 2-APB (Cayman, Ann Arbor, MI) + fractalkine (AF), and 2-APB (A). Cells were treated with 10 nmol/L fractalkine in groups F and AF, 2-APB (50 μmol/L for 1 h) prior to 10 nmol/L fractalkine addition in group AF, serum-free medium in group C, or 50 μmol/L 2-APB in group A.

Wang A., Yang T., Zhang L., Jia L., Wu Q., Yao S., Xu J, & Yang H. (2018). IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 8804-8811.

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Calcium Signaling Reagents Protocol

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All reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA), except that fura-2 acetoxymethyl ester (Fura-2AM) was from Anaspec (Fremont, CA, USA), d-calcium pantothenate was from Fisher Scientific (Pittsburgh, PA, USA), [Arg8] vasopressin (AVP) from American Peptide Co. (Sunnyvale, CA, USA) and 2-APB from Cayman Chemical Co. (Ann Arbor, MI, USA).

Tran T.D., Yao S., Hsu W.H., Gimble J.M., Bunnell B.A, & Cheng H. (2015). Arginine vasopressin inhibits adipogenesis in human adipose-derived stem cells. Molecular and cellular endocrinology, 406, 1-9.

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Cardiomyocyte Isolation and Analysis

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U46619, SQ29548, and 2-APB were purchased from Cayman Chemical (Ann Arbor, MI). Hanks balanced salt solution (HBSS) and Fura-2 AM were obtained from Invitrogen (Carlsbad, CA). Enzymes for cardiomyocyte digestion were purchased from Worthington Biochemical (Lakewood, NJ). Total RNA Isolation kits were purchased from IBI Scientific (Peosta, IA), and the real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using a TaqMan RNA-to-CT 1 step kit and primers and probes from ABI (Carlsbad, CA). Primary antibody for TXA2R was purchased from Abcam (Cambridge, MA). Gentamicin and fetal bovine serum was obtained from Sigma-Aldrich (St. Louis, MO). DeadEnd Fluorometeric TUNEL stain was purchased from Promega (Madison, WI). All remaining reagents were purchased from Fisher Scientific.

Touchberry C.D., Silswal N., Tchikrizov V., Elmore C.J., Srinivas S., Akthar A.S., Swan H.K., Wetmore L.A, & Wacker M.J. (2014). Cardiac thromboxane A2 receptor activation does not directly induce cardiomyocyte hypertrophy but does cause cell death that is prevented with gentamicin and 2-APB. BMC Pharmacology & Toxicology, 15, 73.

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Screening of 2-APB analogues

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All reagents, including the 2-APB analogues, were obtained from Sigma Aldrich (Poole, Dorset, UK) unless otherwise stated. A23187 was from Acros Organics (Fisher Scientific, UK). 2-APB was from Cayman Chemicals (Cambridge Bioscience, Cambridge, UK). SAR-7334 and SN-6 were from Tocris (Bristol, UK). Calcein and BCECF were from ThermoFisher Scientific. Cal-520 was from AAT Bioquest (Sunnyvale CA, USA).

Wei H., Davies J.E, & Harper M.T. (2020). 2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets. Cell Death Discovery, 6, 10.

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Evaluating Anti-seizure Drugs in Rats

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Rats implanted with ventral hippocampal electrodes and dorsal hippocampal optodes were used in these studies. Rats were reused for several drug experiments allowing at least two days between drug treatments. Celecoxib, SC-560, ibuprofen, chelerythrine chloride, milrinone, sildenafil, SKA-31, CAY-10526, seratrodast, ozagrel, paxilline, 2-APB, levetiracetam, and topiramate were obtained from Cayman Chemicals (Ann Arbor, MI). Acetaminophen, nifedipine, bumetanide, ethosuximide, phenytoin, and valproic acid were obtained from Sigma-Aldrich. Lamotrigine was obtained from SelleckChem (Houston, TX). Fasudil was obtained from LC laboratories (Woburn, MA) and phenobarbital was obtained from Strathcona Prescription Center (Canada). Lipophilic drugs were dissolved in 100% DMSO, while hydrophilic drugs were dissolved in saline and injected 30 min prior to seizure induction. The seizure duration and severity of hypoxia (area below 10 mmHg) were compared across kindle (seizure without injection), vehicle-, and drug-treated groups using a within-subject ANOVA and follow-up t-test between vehicle- and drug-treated groups.

Farrell J.S., Gaxiola-Valdez I., Wolff M.D., David L.S., Dika H.I., Geeraert B.L., Rachel Wang X., Singh S., Spanswick S.C., Dunn J.F., Antle M.C., Federico P, & Teskey G.C. (2016). Postictal behavioural impairments are due to a severe prolonged hypoperfusion/hypoxia event that is COX-2 dependent. eLife, 5, e19352.

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Quantifying Protein Levels Using Western Blotting

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Antibodies against STIM1 (1:1000), Myosin II (1:1000) and phospho-Ser19 Myosin light chain II (1:1000) were purchased from Cell Signaling Technology. Antibody against β-actin (clone AC-15) (1:5000) was purchased from Sigma-Aldrich. Antibodies against MMP2 (1:1000) and MMP9 (1:1000) were purchased from Millipore. Antibodies against MT1-MMP (1:500) and cortactin (1:100) were purchased from Santa Cruz Biotechnology. Thapsigargin, SKF-96365, and 2-APB were from Cayman Chemical. For immunoblotting, cells were harvested with ice-cold modified radioimmune precipitation assay (RIPA) buffer containing a protease inhibitor mixture (Roche Diagnostics), 100 mM KCl, 80 mM NaF, 10 mM EGTA, 50 mM h-glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mM vanadate, 0.5% sodium deoxycholate, and 1% NP40. Protein concentrations were determined with the use of a Bio-Rad protein assay. Equal amounts of protein lysates were separated by SDS-PAGE, and then transferred to nitrocellulose blotting membranes (Pall). Immunoblots were blocked, incubated with the primary antibody, washed, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch), and visualized by Western blotting luminol reagent (Santa Cruz Biotechnology). Bands in the immunoblots were quantified using Vision WorksLS software (UVP).

Chen Y.W., Lai C.S., Chen Y.F., Chiu W.T., Chen H.C, & Shen M.R. (2017). STIM1-dependent Ca2+ signaling regulates podosome formation to facilitate cancer cell invasion. Scientific Reports, 7, 11523.

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Fractalkine-Induced Neuroinflammation Modulation

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All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were approved by Animal Ethics and Welfare Committee of Inner Mongolia Autonomous Region People’s Hospital (IACUC-20160427).
Animals were maintained under controlled conditions (24±2ºC, 50–60% humidity, 12/12-h dark/light cycles) with free access to food and water. Anti-mouse CX3CR1 (eBioscience, San Diego, CA) is a neutralizing antibody for CX3CR1, 2-APB (Cayman, Ann Arbor, MI) is an IP₃R antagonist, and SB203580 (Sigma-Aldrich, St. Louis, MO) is a p38MAPK inhibitor. Following complete anesthesia with propofol (i.v. 30 μg/g), we performed a right lateral ventricle puncture according to the Ge ZJ method to deliver reagents directly into the cerebrospinal fluid. All reagents were diluted in 5 μL of sterile normal saline.
At total of 138 adult male C57BL/6 mice, weighing 25–30 g, were randomized and divided into 6 groups: sham (no i.c.v. injection), n=20; vehicle (i.c.v. injection of 5 μL normal saline), n=23; fractalkine (i.c.v. injection of 100 ng fractalkine), n=35; fractalkine + 1 μg anti-CX3CR1 (i.c.v.), n=20; fractalkine + 3.4 μg 2-APB (i.c.v.), n=20; and fractalkine + 1 μg SB203580, n=20. Pretreatments (i.c.v.) were conducted 1 h before i.c.v. injection of 100 ng fractalkine in the last 3 groups.

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Calcium Signaling Experiments in Cells

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BSA, EGTA and reagents for producing Ca²⁺ solutions were from Amresco (Solon, OH, USA). Fetal bovine serum (FBS) was purchased from Bovogen Biologicals (East Keilor, Melbourne, Australia) and heat inactivated at 56 °C for 30 min before use. 2-APB, U-73122 and UDP were from Cayman Chemical (Ann Arbor, MI, USA). Primers for RT-PCR were from Integrated DNA Technologies (Coralville, IA, USA). 5-BDBD, ADP (pre-treated with hexokinase as per [41 (link)]), ATP, BAPTA-AM, BzATP, hexokinase from Saccharomyces cerevisae, ivermectin, MEM non-essential amino acid solution, paraformaldehyde, paroxetine, phosphate buffered saline (PBS), poly-_D-lysine hydrobromide (5 µg∙mL⁻¹ working stock), pluronic F-127, saponin, suramin and UTP were from Sigma-Aldrich (St. Louis, MO, USA). DMEM/F12 medium, ExoSAP-IT, Fura-2 AM, GlutaMAX, penicillin-streptomycin and 0.05% trypsin-EDTA were from ThermoFisher Scientific (Melbourne, Australia). 2MeSADP, AR-C118925, thapsigargin and TNP-ATP were from Tocris Bioscience (Bristol, UK).

Sophocleous R.A., Miles N.A., Ooi L, & Sluyter R. (2020). P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells. International Journal of Molecular Sciences, 21(22), 8572.

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