Methanol and glacial acetic acid were purchased from Mallinckrodt (Phillipsburg, NJ). KGAILMGAILR was synthesized by CPC Scientific (San Jose, CA). Substance P was synthesized by Bachem (Bubendorf, Switzerland). Anti-inflammatory peptide I was synthesized by AnaSpec (Fremont, CA). GLSDGEWQQVLNVWGK was synthesized by SynPep (Dublin, CA). ARACAKA, ARAWAKA, and GRGMGRGMGRL were synthesized by Pepnome Limited (Shenzhen, China). ARAMAKA was synthesized by NeoBioLab (Cambridge, MA). Angiotensin II and sodium periodate were purchased from Sigma Aldrich (St. Louis, MO). All peptide stock solutions for positive nanoelectrospray were prepared in a 49.5/49.5/1 (v/v/v) solution of Methanol/water/acetic acid at an initial concentration of ~1 mg/mL and diluted 100-fold prior to use. The periodate solution was prepared in a 50/50 (v/v) solution of Methanol/water at a concentration of ~1 mg/mL and diluted 10-fold prior to use. For all solution-phase oxidations 5 μL of the prepared periodate solution was added to an equivalent volume of peptide solution.
Substance p
Manufactured by Bachem
Sourced in Switzerland
Substance P is a laboratory product manufactured by Bachem. It is a neuropeptide that functions as a neurotransmitter or neuromodulator. Substance P is involved in the transmission of pain signals and the regulation of various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human tnf, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Trehalose, by Merck Group (2 mentions)
Poly 1 c, by Merck Group (2 mentions)
Sodium periodate, by Merck Group (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Methanol, by Mallinckrodt (1 mentions)
Glacial acetic acid, by Mallinckrodt (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Cell proliferation kit 2, by Roche (1 mentions)
Recombinant mouse il 6, by R&D Systems (1 mentions)
αcgrp, by Bachem (1 mentions)
Anti mouse cd28 mab, by BD (1 mentions)
Compound 48 80 c48 80, by Merck Group (1 mentions)
5 protocols using substance p
1
Peptide Synthesis and Oxidation
2
Inflammatory Stimulus Preparation
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Stock concentrations of LPS (1 mg/mL, Sigma, St. Louis, MO, US), poly(I:C) (10 mg/mL, Sigma), recombinant human TNF (100 µg/mL, ThermoFisher, Waltham, MA, United States), Trehalose (250 mM, Sigma) and AME-1 (diluted 1:2) were prepared in sterile phosphate buffered saline (PBS, Gibco) pH 7.4 without calcium or magnesium. Substance P (Bachem, Bubendorf, CH) was prepared in 0.1 M acetic acid to a concentration of 1 mg/mL. Working stocks of treatments were diluted in culture medium prior to addition to cells.
3
Evaluating Cell Metabolic Activity
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HMC3 were seeded at 0.1 × 105 cells/mL in a 96-well plate and incubated in a humidified atmosphere of 5% CO2 in air at 37°C overnight to adhere. The cells were then treated with AME-1 (50–5,000 µg/mL), poly(I:C) (0.5 µg/mL) (Sigma), LPS (1 µg/mL; Sigma), substance P (0.5 µg/mL) (Bachem), recombinant human TNF (0.25 µg/mL; ThermoFisher), trehalose (0.15–15 mM (Sigma), or untreated for 24 h. Metabolic activity was analysed using the Cell Proliferation Kit II (XTT Assay, Roche, Basel, Switzerland) according to the manufacturer’s instructions and the results are presented as percent metabolic activity relative to untreated cells.
4
Investigating CGRP Signaling Pathways
5
Mast Cell Histamine Release Assay
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The HRA was performed according to a method routinely employed in our laboratory [34 (link),35 (link)]. In brief, MCs pretreated with or without inhibitors were stimulated by FcεRI-aggregation. The anti-AER-37 antibody (eBioscience, San Diego, CA, USA) was used for cultured MCs at 0.1 µg/mL, and the anti-FcεRIα-Ab 29C6 (kind gift from Dr. Hakimi, Hoffmann La Roche, Nutley, NJ, USA) at 0.5 µg/mL served for the stimulation of ex vivo MCs. MRGPRX2 stimulation was achieved by compound 48/80 (c48/80, Sigma, at 10 µg/mL), or substance p (SP, Bachem, Budendorf, Switzerland at 30 µM). Spontaneous release was determined in the absence of any stimulus. Assays were performed in PAG-CM buffer (Piperazine-N,N-bis [2-ethanesulfonic acid]-Albumin-Glucose buffer containing 3 mM CaCl2 and 1.5 mM MgCl2, pH 7.4) for 30 min at 37 °C. Histamine in the supernatants was measured by an automated fluorescence method (Alliance Instruments, Salzburg, Austria). Total cellular histamine content was measured analogously. All determinations were performed in triplicate. Net histamine release (%) was calculated as [(stimulated release–spontaneous release)/complete histamine in the MC preparation] × 100.
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