Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Colonic tissue and cell preparation.
Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed.
Colonic tissue and cell preparation.
GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
Glucose-stimulated GLP-1 secretion in vivo.
Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 µL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO 2 inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for...
Ca 2+ imaging.
Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 min at 37°C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40 × oil-immersion objective. Fura2 was excited at 340/6 and 380/4 nm and Venus at 475/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfu...
FFAR2 and FFAR3 affect GLP-1 release in vivo.
ffar2 and ffar3 knockout mice have impaired glucose tolerance. A: Glucose stimulated GLP-1 secretion in vivo. ffar2 -/- and ffar3 -/- mice and wild-type littermates ( n = 5 each) were dosed with DPP4 inhibitor at a dose of 20 mg/kg per os after a 4-h fast. Thirty minutes post-DPP4 inhibitor dosing, mice were dosed with 1.5 g/kg glucose per os Plasma active GLP-1 was assessed by a MesoScale assay at 0 and 30 min of the oral glucose tolerance test. Data represent means ± 1 SEM, and statistical significance was assessed by Student t test: * P < 0.05 and ** P < 0.01. B - E: Oral glucose tolerance test in ffar2 -/- mice ( n = 8) ( left panel ) and wild-type littermates ( n = 11) ( B and D ) or ffar3 -/- mice ( n = 7) ( right panel ) and wild-type littermates ( n = 6) ( C and E ). Following an overnight fast, mice were given...
Measurement outputs
What raw and processed outputs should exist?
Microscopy-based tissue or cell-state imaging
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Microscopy-based tissue or cell-state imaging.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (5)
Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed.
GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 µL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO 2 inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for active GLP-1 (MesoScale, Gaithersburg, Maryland).
Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 min at 37°C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40 × oil-immersion objective. Fura2 was excited at 340/6 and 380/4 nm and Venus at 475/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfused at ∼1 mL/min. Fura2 fluorescence measurements were taken every 2 s, background corrected, and expressed as the 340:380 nm ratio. Average fluorescence ratios from individual cells were determined over 20-s periods before addition and during perfusion of a test agent. Peak responses to a tes...
ffar2 and ffar3 knockout mice have impaired glucose tolerance. A: Glucose stimulated GLP-1 secretion in vivo. ffar2 -/- and ffar3 -/- mice and wild-type littermates ( n = 5 each) were dosed with DPP4 inhibitor at a dose of 20 mg/kg per os after a 4-h fast. Thirty minutes post-DPP4 inhibitor dosing, mice were dosed with 1.5 g/kg glucose per os Plasma active GLP-1 was assessed by a MesoScale assay at 0 and 30 min of the oral glucose tolerance test. Data represent means ± 1 SEM, and statistical significance was assessed by Student t test: * P < 0.05 and ** P < 0.01. B - E: Oral glucose tolerance test in ffar2 -/- mice ( n = 8) ( left panel ) and wild-type littermates ( n = 11) ( B and D ) or ffar3 -/- mice ( n = 7) ( right panel ) and wild-type littermates ( n = 6) ( C and E ). Following an overnight fast, mice were given 1.5 g/kg glucose per os, and blood glucose ( B and C ) and insulin ( D and E ) were measured at the time points indicated. F and G: Insulin tolerance test in ffar2 -/- mice ( n = 11) ( left panel ) and wild-type littermates ( n = 6) ( F ) or in ffar3 -/- mice ( n = 7) ( rig...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods",
"description": "Protocol for Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Colonic tissue and cell preparation.",
"text": "Mice aged 6-26 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described ( ). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Colonic tissue and cell preparation.",
"text": "GLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol/L glucose) supplemented with 10% FBS, 2 mmol/L L -glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Glucose-stimulated GLP-1 secretion in vivo.",
"text": "Experiments were performed on 3- to 4-month-old ffar2 -/- and ffar3 -/- mice or wild-type littermate control 129/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 µL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO 2 inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Ca 2+ imaging.",
"text": "Experiments were performed 4-7 days postplating, using colonic tissue from GLU-Venus transgenic mice with L cell-specific Venus expression ( ). Cells were loaded with 7 µmol/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 µmol/L eserine in standard saline solution for 30 min at 37°C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40 × oil-immersion objective. Fura2 was excited at 340/6 and 380/4 nm and Venus at 475/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfu..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "FFAR2 and FFAR3 affect GLP-1 release in vivo.",
"text": "ffar2 and ffar3 knockout mice have impaired glucose tolerance. A: Glucose stimulated GLP-1 secretion in vivo. ffar2 -/- and ffar3 -/- mice and wild-type littermates ( n = 5 each) were dosed with DPP4 inhibitor at a dose of 20 mg/kg per os after a 4-h fast. Thirty minutes post-DPP4 inhibitor dosing, mice were dosed with 1.5 g/kg glucose per os Plasma active GLP-1 was assessed by a MesoScale assay at 0 and 30 min of the oral glucose tolerance test. Data represent means ± 1 SEM, and statistical significance was assessed by Student t test: * P < 0.05 and ** P < 0.01. B - E: Oral glucose tolerance test in ffar2 -/- mice ( n = 8) ( left panel ) and wild-type littermates ( n = 11) ( B and D ) or ffar3 -/- mice ( n = 7) ( right panel ) and wild-type littermates ( n = 6) ( C and E ). Following an overnight fast, mice were given..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2",
"datePublished": "2012",
"author": [
{
"@type": "Person",
"name": "Gwen Tolhurst"
},
{
"@type": "Person",
"name": "Helen Heffron"
},
{
"@type": "Person",
"name": "Yu Shan Lam"
},
{
"@type": "Person",
"name": "Helen E. Parker"
},
{
"@type": "Person",
"name": "Abdella M. Habib"
},
{
"@type": "Person",
"name": "Eleftheria Diakogiannaki"
},
{
"@type": "Person",
"name": "Jennifer Cameron"
},
{
"@type": "Person",
"name": "Johannes Grosse"
},
{
"@type": "Person",
"name": "Frank Reimann"
},
{
"@type": "Person",
"name": "Fiona M. Gribble"
}
],
"identifier": "10.2337/db11-1019"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2 methods",
"item": "https://replicatescience.com/experiments/short-chain-fatty-acids-stimulate-glucagon-like-peptide-1-secretion-via-the-g-protein-8211-coupled-receptor-ffar2-methods-gwen-tolhurst-pmc3266401/short-chain-fatty-acids-stimulate-glucagon-like-peptide-1-secretion-via-the-g-protein-coupled-recept-mlpgvl1t"
}
]
}
]