Source Paper
Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein–Coupled Receptor FFAR2
Gwen Tolhurst, Helen Heffron, Yu Shan Lam, Helen E. Parker, Abdella M. Habib et al.
Diabetes • 2011
Glucose-Stimulated GLP-1 Secretion In Vivo
Objective: Measure active GLP-1 secretion in response to glucose gavage in fasted ffar2 and ffar3 knockout mice versus wild-type controls following DPP4 inhibitor pretreatment
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Protocol Steps
Animal fasting
Mice fasted for 4 hours prior to DPP4 inhibitor dosing
Note: Mice aged 3-4 months used
View evidence from paper
“Mice were fasted for 4 h and dosed per os with 20 mg/kg DPP4 inhibitor”
DPP4 inhibitor administration
Oral gavage administration of DPP4 inhibitor (cat. no. KR-62436) at 20 mg/kg
Note: Route: per os (oral gavage)
View evidence from paper
“dosed per os with 20 mg/kg DPP4 inhibitor (cat. no. KR-62436; Sigma)”
Baseline blood collection
Collect 150 µL blood from awake mice via tail bleed into EDTA-coated capillary tubes before glucose dosing
Note: Initial GLP-1 measurement timepoint
View evidence from paper
“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”
Glucose gavage
Thirty minutes after DPP4 inhibitor dosing, administer glucose solution by oral gavage at 1.5 g/kg
Note: Route: oral gavage
View evidence from paper
“Thirty minutes later, mice were dosed by gavage with 1.5 g/kg glucose solution (Fisher Scientific)”
Terminal blood collection
Thirty minutes after glucose dosing, kill mice by CO2 inhalation and collect blood via cardiac puncture into aprotinin-containing tubes
Note: Euthanasia method: CO2 inhalation. Blood collected into tubes containing aprotinin (0.6 trypsin inhibiting units/mL)
View evidence from paper
“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)”
Plasma preparation
Centrifuge blood samples immediately and freeze plasma on dry ice
Note: Samples prepared for active GLP-1 assay
View evidence from paper
“Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for active GLP-1”
Active GLP-1 assay
Assay plasma samples in duplicate for active GLP-1 using MesoScale ELISA
Note: Samples assayed in duplicate
View evidence from paper
“plasma was frozen on dry ice before assay in duplicate for active GLP-1 (MesoScale, Gaithersburg, Maryland)”