In both the ICV study and the intra-VTA study injections were performed according to a cross-over balanced experimental design, in which all rats at one point received an injection of either ghrelin or artificial cerebrospinal fluid (aCSF; Tocris, Bristol, U.K) separated with one day wash out period. This corresponds to days 15 and 17 of exposure to the choice diet. All the injections were performed over a 1 min period using a 10 μl Hamilton syringe coupled to an injector needle via vinyl tubing. After injection the injector was kept in place for approximately 2 min to ensure full diffusion from the injector tip. The injected volume was 2 μl into the lateral ventricle, and 1 μl unilaterally into the VTA. ghrelin (1465; Tocris, Bristol, UK) and NPY (H-6375; Bachem, Bubendorf, Switzerland) were dissolved in aCSF. ghrelin was injected at a dose of 2 μg ICV, and 1 μg intra-VTA, doses that previously has been shown to induce a feeding response in rats [16 (link), 20 (link)]. For intra-VTA injection, we used coordinates that correspond to the published distribution of the ghrelin receptor (GHS-R1A)[21 (link)] and used a 1μl delivery volume in order to ensure that we reached the entire VTA with the injected substance.
Ghrelin
Manufactured by Bio-Techne
Sourced in United Kingdom
Ghrelin is a lab equipment product manufactured by Bio-Techne. It is a peptide hormone that is responsible for regulating hunger and growth hormone release.
Automatically generated - may contain errors
Lab products found in correlation
Cck octapeptide, by Bachem (2 mentions)
Pyy 3 36, by R&D Systems (2 mentions)
D lys3 ghrp 6, by Bio-Techne (2 mentions)
D lys3 ghrp 6, by Apexbio (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Apomorphine, by Merck Group (1 mentions)
Rat ghrelin, by Peptide Institute (1 mentions)
Act462206, by Bio-Techne (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Cck 8 assay, by Apexbio (1 mentions)
Lps o127 b8, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BestBio (1 mentions)
Matrigel growth factor reduced basement membrane matrix, by Corning (1 mentions)
Foetal bovine serum (fbs), by Sartorius (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Acylated ghrelin, by Bachem (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Biie0246, by Bio-Techne (1 mentions)
15 protocols using ghrelin
1
Ghrelin Administration in Rat Feeding Behavior
2
Cultured Mouse Kidney Collecting Duct Cells
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A mouse kidney cortical collecting duct (mpkCCD) cell line was maintained in Dulbecco’s modified Eagle’s medium consisting of Ham’s F12 medium (Gibco, Carlsbad, CA), 5% foetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), and 1% penicillin/streptomycin (Corning Life Sciences, Tewksbury, MA) at 37 °C with 5% CO2 and 95% air. Ghrelin and the Ghrelin receptor antagonist [D-Lys3]-GHRP-6 were both purchased from Tocris Bioscience (Bristol, UK).
3
Ghrelin and GHSR Antagonist Study
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Ghrelin was purchased from Tocris Bioscience (No. 1463/1). [D-Lys3]-GHRP-6 (GHSR antagonist) was purchased from APEXBIO (No. B5234). Pontamine sky blue (C8679) and glucose (G7528) were purchased from Sigma-Aldrich (for electrophysiology experiment). 0.9% NaCl and glucose were obtained from Qingdao University hospital. The chloral hydrate was purchased from Tianjin Ruijinte chemical company. All other chemicals are of the highest grade obtainable.
4
Ghrelin and Tamoxifen Administration
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5
Ghrelin, Leptin, and Apomorphine Regulation
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Ghrelin (250 μg kg−1; Tocris, Bristol, UK), leptin (1 mg/kg; National Hormone and Peptide Program (NHPP), USA) as well as vehicle (saline) were injected i.p. 5 min before recordings started. These concentration were previously shown to affect food intake and neuronal activity.28 (link) Saline was injected in an identical volume (0.1 ml per 100 g) to control for neuronal responses to the injection. Apomorphine (0.1 mg kg−1; Sigma-Aldrich, Saint Louis, MO, USA) was administered i.p. 30 min following task execution, to identify DA neurons.
6
Intracerebroventricular Injection of Novel DORA and Ghrelin
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For the icv injection of ACT462206, ghrelin, and saline, a stainless steel injector (300-µm, outer diameter) was introduced through the cannula at a depth of 1.0 mm beyond the end of the guide cannula. The total volume of the solutions of ACT462206 (or saline) and ghrelin (or saline) injected into the lateral ventricle was 5 µL. A novel DORA, ACT462206, [(2S)-N-(3,5-dimethylphenyl)-1-[(4-methoxyphenyl)sulfonyl]-2-pyrrolidinecarboxamide] was purchased from TOCRIS Bioscience (Bristol, UK). Rat ghrelin was purchased from the Peptide Institute (Minoh, Japan). ACT462206 and ghrelin were dissolved in sterile 0.9% saline.
7
Ghrelin-Induced Food Intake in Mice
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ghrelin-induced food intake was assessed in adult wild-type (n = 9), heterozygous (n = 9), and homozygous (n = 6) Ghsr-IRES-Cre mice. Mice received subcutaneous (s.c.) injections of saline for three consecutive days for habituation to the procedure. Injections were performed in a cross-over fashion: every animal at one point received either ghrelin or an equal volume saline vehicle one day apart. On the experimental day, food was withdrawn from the cages during the light period (9 am) for 3 h, after which ghrelin (3 mg/kg BW s.c.; #1465; Tocris, Bristol, UK) or saline was administered. Immediately after injection, pre-weighed regular chow was re-introduced on the cage floor. Food intake was manually measured at 3 and 24 h post-ghrelin administration using calibrated scales that had a precision of 1 mg. Mice were also weighed 24 h post-ghrelin administration in order to determine the transient impact of peripherally administered ghrelin on weight gain.
8
Neurotransmitter Dosing in Rodents
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Ghrelin (Tocris, 1465), CCK octapeptide (Bachem, CAT: 4033010), PYY 3–36 (R&D Systems, Cat#1618), and 5-hydroxytryptamine hydrochloride (5-HT; Sigma-Aldrich, Cat#H9523), were dissolved in saline at stock concentrations and stored at −80°C until use. On the day of testing, the stock was thawed, diluted with saline, and delivered at a volume of 10 ml/kg at the following doses: Ghrelin (1 mg/kg), CCK (10 μg/kg), 5-HT (2 mg/kg), and PYY (0.1 mg/kg).
9
Ghrelin Modulates Cell Viability and Apoptosis
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Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) was used to culture cells. LPS (O127:B8) obtained from Sigma Chemical Co was dissolved in distilled water to a final concentration of 1 μg/ml. Ghrelin was purchased from Tocris (cat. no. 1463; Tocris) and was formulated into a solution with a concentration of 0.1 nmol/ml. CCK-8 assays (APExBIO) were used to determine cell viability. TUNEL assays (KeyGen Biotech Co, Ltd) and an Annexin V-FITC/PI apoptosis detection kit (BestBio) were also used. Growth factor–reduced Matrigel Basement Membrane Matrix was purchased from Corning Co. All antibodies, secondary Alexa Fluor 594–conjugated goat anti-rabbit IgG, and the PI3K inhibitor LY294002 were purchased from Cell Signaling Technology.
10
Nutrient Deprivation Modulates Cell Signaling
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Cells were exposed to nutrient deprivation to mimic a caloric restriction condition, Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) low glucose medium (1 g.L−1 glucose, 100 U.mL−1 penicillin and 100 μg.mL−1 streptomycin, without B27 supplementation), NPY (100 nM; Phoenix Europe GmbH, Karlsruhe, Germany) or acylated ghrelin (10 nM; Bachem, Bubendorf, Switzerland). Cells were also exposed to the lysosomal protein degradation inhibitor, chloroquine (100 μM; Sigma-Aldrich), NPY receptors selective antagonists (all at 1 μM; all from Bachem, Bubendorf, Switzerland; NPY Y1 antagonist (BIBP3226), NPY Y2 antagonist (BIIE0246) and NPY Y5 antagonist (L-152,804)), and/or ghrelin receptor antagonist ([D-Lys3]-GHRP-6) (100 μM; Tocris Bioscience, Bristol, UK), added to the cell culture medium 30 min prior to caloric restriction, NPY or ghrelin treatment for 6 h.
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