The peptide SLIGRL and the TRPA1 antagonist HC-030031 were purchased from Sigma-Aldrich Chemicals, Co. (St. Louis, MO, USA). SLIGRL was dissolved in saline to obtain 7.5, 15, and 40 mM/1 μL final dose, and HC-030031 was dissolved in 30 μL 1% DMSO and saline to obtain final dose 50 and 100 μg/30 μL. Various doses of this chemical were injected intra-plantar through a 30G needle connected by PE 50 tubing to a Hamilton micro-syringe. The same volumes of vehicle (isotonic saline) were microinjected in the same manner separately as a control. In the second set of experiments, 15–20 min prior to the start of the experiment, the same volume of the TRPA1 antagonist HC-030031 was pre-injected in the same hindpaw and animals were examined by the thermal and mechanical paw tests. Different animal groups were used for the experiments and they were tested with one concentration of irritant chemicals, antagonists, or vehicle, and not repeatedly used. Six mice were used for each group.
Sligrl
Manufactured by Merck Group
Sourced in United States
SLIGRL is a synthetic peptide used in laboratory research. It functions as a selective agonist for the protease-activated receptor 2 (PAR2). This receptor is involved in various biological processes and is a target of interest in scientific investigations.
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Lab products found in correlation
Hc 030031, by Merck Group (1 mentions)
C48 80, by Merck Group (1 mentions)
A23187, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Recombinant egf, by R&D Systems (1 mentions)
Myc clone 9e10, by Santa Cruz Biotechnology (1 mentions)
Complete dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Endothelin 1, by Merck Group (1 mentions)
Histamine, by Merck Group (1 mentions)
Par2 ap, by Merck Group (1 mentions)
5 protocols using sligrl
1
Examining TRPA1 Antagonist Effects
2
Oligonucleotide Synthesis and Characterization
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Synthetic, endotoxin-free, and completely phosphorothioate-modified oligonucleotides, denoted ssON and ON15, were purchased from Integrated DNA Technologies. ssON has the following sequence: 5′-GAAGTTTTGAGGTTTTGAAGTTGTTGGTGGTGGTG-3′. ON15 has the following sequence: 5′-GGTTTTGAAGTTGTT-3′. C48/80, SLIGRL, SP and A23187 were purchased from Sigma-Aldrich. LL-37 was purchased from Bachem and Anaspec.
3
Primary Bronchial Epithelial Cell Culture
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Primary human bronchial epithelial cells were obtained from American Type Culture Collection and cultured in airway epithelial medium (American Type Culture Collection). HEK293T and COS-7 cells were cultured in complete Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (ThermoFisher). CCh, MCh, vecuronium, SLIGRL, and LPS were purchased from Sigma. Recombinant EGF, IL-1β, TGFβ, and TNFα were from R&D Systems. Antibodies were purchased from the following sources: RGS4 (catalog no.: ABT17) and β-actin were from Sigma Millipore; p85α (Cell Signaling Technology); and Myc (clone 9E10) and GFP were from Santa Cruz Biotechnology. Recombinant 6His-p85α was purchased from MyBioSource, and 6His-lyn kinase was from Sigma.
4
Pruritogen-Induced Scratching Behavior in Mice
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At least 24 h before testing, mice were shaved at the nape of the neck under isoflurane anesthesia. The following pruritogens were dissolved in saline and injected (50 μl, s.c.) into the neck: chloroquine (Sigma, 100 μg), endothelin-1 (Sigma, 25 ng), histamine (Sigma 500 μg), SLIGRL (Sigma, 100 μg), and thymic stromal lymphopoietin (TSLP; Sigma, 2.5 μg). The mice were video recorded, and from these videos, we counted the total number of discrete scratching bouts that occurred during the first 30 min after injection.
5
Isolation and Quantification of Microvesicles
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Cells (2 × 105) were seeded out in 6-well plates and permitted to adhere. All cells lines were washed and pre-adapted to respective serum-free medium prior to activation and harvesting of conditioned media. To induce microvesicle release, the cells were stimulated with PAR2-activating peptide (PAR2-AP); SLIGRL; (20 μM) (Sigma). The released cell-derived microvesicles were then isolated from conditioned media and resuspended in PBS according to published procedures [24 (link), 25 (link), 32 (link)–34 ]. The microvesicles were quantified using the Zymuphen MP assay kit (Hyphen BioMed/Quadratech Ltd, Epsom, UK) since this attribute was relevant to the functionality of the microvesicles and consequently, to this study.
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