Primary cells were isolated from rat pituitary as previously described (Kojima et al. 1999 (link)). Primary pituitary cells were stimulated with LEAP2 (1 µM) and/or ghrelin (100 nM). To examine the effect of LEAP2 on GH release in vivo, saline or LEAP2 (15 nmol in 50 µL saline) was introduced through the jugular vein. After 10 min, ghrelin (5 nmol) was administered through the jugular vein, and blood samples were taken from the tail tip at 0, 15, and 30 min. GH was measured using the Rat/Mouse GH ELISA Kit (Millipore EMD).
Rat mouse gh elisa kit
Manufactured by Merck Group
Sourced in Sweden, United States
The Rat/Mouse GH ELISA kit is a quantitative immunoassay designed for the measurement of growth hormone (GH) levels in rat and mouse serum or plasma samples. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify GH concentrations.
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8 protocols using rat mouse gh elisa kit
1
Investigating LEAP2 Effects on Pituitary GH Release
2
Measuring Plasma GH and IGF-1 in Mice
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Plasma levels of GH and IGF-1 were measured in female mice (n = 4‐6) at indicated ages. After overnight fast, blood was collected from the orbital sinus and plasma was separated for the measurement of IGF-1 using the ACTIVE Mouse/Rat IGF-1 RIA kit (Diagnostic Systems Laboratories Inc.). Plasma GH was measured using the Rat/Mouse GH ELISA Kit (Millipore).
3
Plasma IGF-1 and GH Quantification
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Plasma IGF-1 concentration was determined using mouse/rat IGF-1 Quantikine ELISA Kit (R and D Biosystems, Cat #: MG100) without fasting. Mice were fasted for 36 hr before GH stimulation testing. Fifteen minutes after anesthesia with pentobarbital (50 mg/kg given once i.p.), 0.14 g/kg GHRH (Phoenix Pharmaceuticals, Cat #:031–02) was injected i.p. Blood was sampled retro-orbitally using a capillary tube before, 5, and 15 min after injection. Plasma GH concentration was measured using a Rat/Mouse GH ELISA KIT (Millipore, Cat #: EZRMGH-45K).
4
Ghrelin-Induced Growth Hormone Release in Rats
5
Fasting Induces Hormonal Changes
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Blood was collected from mice 6 hr after fasting. Serum insulin and GH levels were measured using the Mercodia mouse insulin ELISA Kit (Mercodia AB, Uppsala, Sweden) and the rat/mouse GH ELISA Kit (EMD Millipore, ON, Canada), respectively. IGF-I levels in the serum or in protein extracts were analyzed using the Mouse/Rat IGF-I Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).
6
Ghrelin-Induced Growth Hormone Release in Rats
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WT (n = 8) and KO (n = 8) rats were challenged with acyl-ghrelin to induce GH release. Rats received counterbalanced IP injections of 0 nmol (saline) or 4.5 nmol/kg ghrelin over two days, with three days of washout in between. The 4.5 nmol/kg dose was chosen because it has been previously shown to stimulate GH release (30 (link)). Tail blood was collected 15 min post IP injection. Blood was collected in EDTA coated tubes and stored on ice until centrifuged at 4000 RPM for 15 min. The supernatant from each sample was then stored at −80 °C until they were assayed. Plasma GH concentrations were analyzed using a commercial rat/mouse GH ELISA kit (EMD Millipore, Billerica, MA) and assayed in duplicate according to the manufacturer’s instructions. The intra- and inter-assay variations for rat GH were less than 5%. GH concentrations were interpolated using the 5-parameter logic equation from standard curve running for each plate.
7
Serum Biomarker Profiling in Rodents
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ELISA was performed to detect serum GH (rat/mouse GH ELISA kit; Merck KGaA), IGF-1 (mouse/rat IGF-1 ELISA kit; R&D Systems), and total thyroxine (total thyroxine ELISA; ALPCO) according to the protocols of the respective ELISA kits. Serum amylase and lipase activities were measured using kits from BioAssay Systems (ECAM-100 and DLPS-100) according to the manufacturer's instructions.
8
ATRA Modulation of Pituitary GH Secretion
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The anterior pituitary cells were seeded into 24-well plates at a concentration of 1 × 10 5 cells/well and exposed to ATRA (10 -6 M) for 72 h in DMEM/F12 medium with 10% dextran-coated charcoal-treated fetal bovine serum. After treatment with ATRA, the cells were washed with DMEM/F12 medium with 0.1% bovine serum albumin (Roche Diagnostics, Mannheim, Germany), after which the media were incubated for 30 min. Media were changed, GHRH or ghrelin was then added to 500 µL of media at a concentration of 10 -9 M and the cells were incubated for 30 min. GH concentrations in the media were measured by a Rat/Mouse GH ELISA Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instruction.
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