Substance p

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Substance P is a neuropeptide that functions as a neurotransmitter and neuromodulator in the central and peripheral nervous systems. It is involved in the transmission of pain signals and the regulation of various physiological processes. Substance P is commonly used in research applications to study its role in neurological and biological processes.

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14 protocols using substance p

CGRP and Substance P Signaling

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CGRP (1 µg/mouse; 1161; Tocris) and substance P (250 µg/kg; 1156; Tocris) were dissolved in PBS and injected i.p. to mice, separately or combined. The Adrb2 selective agonist clenbuterol (5 mg/kg) was i.p. injected to mice daily.

Chen C.S., Weber J., Holtkamp S.J., Ince L.M., de Juan A., Wang C., Lutes L., Barnoud C., Kizil B., Hergenhan S.M., Salvermoser J., Lasch M., Deindl E., Schraml B., Baumjohann D, & Scheiermann C. (2021). Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ. The Journal of Experimental Medicine, 218(8), e20202377.

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Neurochemical Profiling of Dopamine Signaling

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Atropine sulfate, guanethidine monosulphate, N^ω-nitro-L-argininemethylester, 6-hydroxy dopamine, and ascorbic acid were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Tetrodotoxin, Substance P, N-acetyl-L-tryptophan 3,5-bis (trifluoromethyl) benzyl ester, GR159897, and SB218795 were purchased from Tocris (Bristol, UK). Mouse anti-TH antibody (1:2000) was purchased from Chemicon International (Temecula, CA, USA). Biotinylated anti-mouse IgG antibody (1:1000) and nickel-intensified 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit for peroxidase) were purchased from Vector Laboratories (Burlingame, CA, USA). Neutral-buffered formaldehyde (NBF) and xylene were purchased from Carlo Erba (Milan, Italy).

Pellegrini C., Fornai M., Colucci R., Tirotta E., Blandini F., Levandis G., Cerri S., Segnani C., Ippolito C., Bernardini N., Cseri K., Blandizzi C., Haskó G, & Antonioli L. (2016). Alteration of colonic excitatory tachykininergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration. Journal of Neuroinflammation, 13, 146.

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Substance P Regulates Vascular Endothelial Cell Growth

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VECs (5 × 10⁴ cells / well in a 12-well plate) were co-cultured with TG neurons or cultured in the TG conditioned media for 24 hours. To determine the role of SP in VEC growth, 2μM spantide I (Tocris Bioscience, Minneapolis, MN, USA) was added to the conditioned media. The cell numbers of VEC were counted directly using a hemacytometer under a 20× objective phase contrast microscope field. Each sample was counted in four separate fields and the mean cell number was used for analysis. The assay was repeated twice. The effect of substance P on VEC growth was evaluated with BrdU Cell Proliferation Kit (MilliporeSigma, Cat. No 2752, Burlington, MA, USA).VEC (5 × 10³ cells / well) were seeded in a 96-well plate. Serum-starved VECs were cultured in the presence or absence of 1μM substance P (Tocris Bioscience) with DMEM supplemented with 1% FBS for 24 hours. Proliferating cells were labeled with BrdU during the last 22 hours of the culture period. BrdU incorporation into the cells was quantified by the absorbance value at 450 nm. The assay was repeated three times.

Liu L., Dana R, & Yin J. (2020). Sensory neurons directly promote angiogenesis in response to inflammation via substance P signaling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(5), 6229-6243.

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Pharmacological Preparations for Neurophysiology

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[D-Ala², NMe-Phe⁴, Gly-ol⁵]-enkephalin (DAMGO) and substance P were from Tocris (Ellisville, MO). Other chemicals were from Sigma. Drugs were prepared as stock solutions of 10–100 mM in the appropriate solvent and then diluted in aCSF. Thiorphan was dissolved in DMSO; other compounds were dissolved in water.
Artificial cerebrospinal fluid (aCSF) contained (in mM) 124 NaCl, 1.9 KCl, 26 NaHCO₃, 1.2 KH₂PO₄, 1.3 MgSO₄, 2.4 CaCl₂ and 10 glucose, and was gassed with 95% O₂ / 5% CO₂. Sucrose-aCSF was the same medium with 5 mM KCl and 215 mM sucrose instead of NaCl. K⁺-aCSF was aCSF containing 5 mM KCl.

Chen W., McRoberts J.A, & Marvizón J.C. (2014). μ-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain. Neuroscience, 267, 67-82.

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Neurotransmitter Regulation of NGF Signaling

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Drugs were dissolved in HBSS, which was used as the experimental vehicle, and applied directly to the media containing NGF. Drugs concentrations are based on published literature: choline (Acros Organics, Geel, Belgium) (10 mM, 3 mM, and 1 mM) [22 (link)]; α-bungarotoxin (Thermofisher) (BGTX) (50 nM) [32 (link)]; calpeptin (Sigma Aldrich, St. Louis MO, USA)(26 μM) [33 ], ryanodine (Santa Cruz) (30 μM) [21 (link)]; Xestospongin C. (Tocris Biosciences, Bristol, UK.) (Xest C.) (1 μM) [20 (link)]; FK506 (Tocris Biosciences) (40 μM) [34 (link)]; Substance P (Tocris Biosciences) (Sub P) (1 μM) [20 (link)]; PNU 282987 (Tocris Biosciences) (10 μM) [35 (link)].

King J.R, & Kabbani N. (2018). Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone. PLoS ONE, 13(5), e0197247.

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Neuropeptide Effects on B Cell Activation

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Splenic B cells were isolated from naive C57BL/6 mice by following the instructions of an EasySep Mouse B Cell Isolation Kit (STEMCELL Technologies 19854) and cultured at density of 2 × 10⁶ cells/well, 1 mL/well, in 24-well plates. Stimulation media were prepared by adding IL-4 (BioLegend 574306; 20 ng/mL) and LPS (MilliporeSigma L-4391; 10 μg/mL) in RPMI supplemented with FB Essence (Avantor Seradigm 10803-034; 10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) (Corning 30-002-CI as mixed penicillin/streptomycin solution), and β-mercaptoethanol (Gibco 21985023; 55 μM). Neuropeptides αCGRP (Bachem AG 4025897.0500; 300 nM) or substance P (Tocris 1156; 1 μM) were added to the indicated groups. After 96-hour culture, supernatant samples were collected for ELISA, and cells were harvested for FACS analysis.

Mathur S., Wang J.C., Seehus C.R., Poirier F., Crosson T., Hsieh Y.C., Doyle B., Lee S., Woolf C.J., Foster S.L, & Talbot S. (2021). Nociceptor neurons promote IgE class switch in B cells. JCI Insight, 6(24), e148510.

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Intradermal Immunization in Mice

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Mice were immunized intradermally (i.d) in the right side of the cheek (behavioral experiments), base of tail (Kaede experiments), or in footpads. As indicated, mice were immunized with 50 µg of papain, heat-inactivated papain, ovalbumin, histamine, or 40 µg of Capsaicin (all from Sigma-Aldrich); or 100 µg of Alternaria alternata (Greer). Where indicated, mice were injected with 10 µg LPS (InvivoGen), 5 µg CGRP (Sigma-Aldrich), 100 nmoles Substance P (Tocris) or 1% QX314 (Tocris). All dilutions were diluted in sterile 1x phosphate buffered saline, or PBS (Corning), except for Capsaicin which was diluted in PBS and 6.4% DMSO. Where indicated, immunizations were performed with either 500 µmol of QWF or L733060 (both from Tocris) diluted in a 5% DMSO PBS solution.

Perner C., Flayer C.H., Zhu X., Aderhold P.A., Dewan Z.N., Voisin T., Camire R.B., Chow O.A., Chiu I.M, & Sokol C.L. (2020). Substance P release by sensory neurons triggers dendritic cell migration and initiates the Type-2 immune response to allergens. Immunity, 53(5), 1063-1077.e7.

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Substance P Receptor Modulation

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Complete Fruend’s Adjuvant was purchased from Sigma, Substance P and SR 140333 from Tocris, and Anti-Substance P Antibody for behavior studies was purchased from Millipore Sigma (AB1566) and isotype IgG was used as control.

Green D.P., Limjunyawong N., Gour N., Pundir P, & Dong X. (2019). A Mast-Cell-Specific Receptor Mediates Neurogenic Inflammation and Pain. Neuron, 101(3), 412-420.e3.

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Substance P Therapy for DSS-Induced Colitis

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Substance P was purchased from Tocris and administered intraperitoneally every other day at dose of 2 μg per mouse starting from day 0 to day 10 during DSS-induced colitis.

Zhang W., Lyu M., Bessman N.J., Xie Z., Arifuzzaman M., Yano H., Parkhurst C.N., Chu C., Zhou L., Putzel G.G., Li T.T., Jin W.B., Zhou J., Hu H., Tsou A.M., Guo C.J, & Artis D. (2022). Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection. Cell, 185(22), 4170-4189.e20.

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Perfusion of Tissues for Imaging and Electrophysiology

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Tissues used for imaging and electrophysiological experiments were perfused with KRB containing (mmol/L): NaCl, 120.35; KCl, 5.9; NaHCO₃, 15.5; NaH₂PO₄, 1.2; MgCl₂, 1.2; CaCl₂, 2.5; and glucose, 11.5. KRB was bubbled with a mixture of 97% O₂ – 3% CO₂ and warmed to 37 ± 0.2 °C. Atropine, neostigmine, N_ω-nitro-L-arginine (L-NNA) and carbachol (CCh) were purchased from Sigma-Aldrich (St Louis, MO, USA). Tetrodotoxin (TTX), Ani9, cyclopiazonic acid (CPA), RP 67580, Substance P, GR 73632, AF-DX 116, DAU 5884, MEN 10376 and MRS 2500 were purchased from Tocris Bioscience (Ellisville, Missouri, USA). GSK 7975A was purchased from Aobious. All drugs were dissolved as recommended by the manufacturers and then diluted to the desired concentrations with KRB solution.

Drumm B.T., Rembetski B.E., Huynh K., Nizar A., Baker S.A, & Sanders K.M. (2020). Excitatory cholinergic responses in mouse colon intramuscular interstitial cells of Cajal are due to enhanced Ca2+ release via M3 receptor activation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(8), 10073-10095.

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