Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice 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?
Methods
Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mice were 9 weeks of age. During the first 5 days of a 3-week adaptation period, mice were fed a standard Harlan chow diet, followed by a standardised semi-synthetic normal-fat diet [NF, 10 energy% (en%) fat] with the same dietary constituents as the intervention high-fat diet (HF, 40 en%) in which carbohydrates were substituted with fats (Hoevenaars et al. ) (Research Diets Services B.V., Wijk bij Duurstede, The Netherlands). At the start of the 12-week intervention period, mice were stratified based on body weight over 7 groups ( n = 12), to obtain ident...
Motor performance
Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run duration of 0.5 s and maximum run duration of 10 s. Quantitative gait parameters were analysed using the CatWalk XT 10.0 software (Noldus Information Technology).
Lipid determination in serum and liver
Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1 H nuclear magnetic resonance ( 1 H-NMR), and neutral lipids were stained in frozen liver sections with Oil red O (Sigma) and quantified.
Statistical analysis
All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurements ( n = 6), subsets of mice as indicated between the brackets were randomly selected, because of limited equipment or practical reasons. GraphPad Prism version 5.03 (GraphPad software, San Diego, CA, USA) was used for statistical analysis. Data were checked for normality and if needed log transformed ( Acot3, Cyp4a14, Por, Fasn and Cyp2b9 in liver and Cpt1a in EpiWAT). One-way ANOVA was used to compare the groups, followed by Dunnett's post hoc test to compare the different HF + flavonoid groups to HF, and HF to NF. For the analysis of data of...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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: Gait recovery and locomotor function.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (4)
Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mice were 9 weeks of age. During the first 5 days of a 3-week adaptation period, mice were fed a standard Harlan chow diet, followed by a standardised semi-synthetic normal-fat diet [NF, 10 energy% (en%) fat] with the same dietary constituents as the intervention high-fat diet (HF, 40 en%) in which carbohydrates were substituted with fats (Hoevenaars et al. ) (Research Diets Services B.V., Wijk bij Duurstede, The Netherlands). At the start of the 12-week intervention period, mice were stratified based on body weight over 7 groups ( n = 12), to obtain identical groups for this important parameter. Male mice were used in order to enable comparison of outcomes with previous findings (Hoek-van den Hil et al. ). One group of mice continued on NF, while the other six groups of mice received HF with or without supplementation of different flavonoids (HF...
Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run duration of 0.5 s and maximum run duration of 10 s. Quantitative gait parameters were analysed using the CatWalk XT 10.0 software (Noldus Information Technology).
Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1 H nuclear magnetic resonance ( 1 H-NMR), and neutral lipids were stained in frozen liver sections with Oil red O (Sigma) and quantified.
All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurements ( n = 6), subsets of mice as indicated between the brackets were randomly selected, because of limited equipment or practical reasons. GraphPad Prism version 5.03 (GraphPad software, San Diego, CA, USA) was used for statistical analysis. Data were checked for normality and if needed log transformed ( Acot3, Cyp4a14, Por, Fasn and Cyp2b9 in liver and Cpt1a in EpiWAT). One-way ANOVA was used to compare the groups, followed by Dunnett's post hoc test to compare the different HF + flavonoid groups to HF, and HF to NF. For the analysis of data of liver weight, hepatic gene expression of Acacb and Cpt1a, and WAT gene expression of Pparg, a Kruskal-Wallis test was used, because also after log transformation these data were not normally distributed. Curve fitting was used to analyse body weight gain during the 12-week intervention period...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice methods",
"description": "Protocol for Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Methods",
"text": "Eighty-four male C57BL/6JOlaHsd mice (Harlan Laboratories, Horst, The Netherlands) were individually housed under controlled conditions (temperature 21 °C, 12 h/12 h light-dark cycle, 55 ± 15 % humidity), with ad libitum access to food and water. At arrival, the mice were 9 weeks of age. During the first 5 days of a 3-week adaptation period, mice were fed a standard Harlan chow diet, followed by a standardised semi-synthetic normal-fat diet [NF, 10 energy% (en%) fat] with the same dietary constituents as the intervention high-fat diet (HF, 40 en%) in which carbohydrates were substituted with fats (Hoevenaars et al. ) (Research Diets Services B.V., Wijk bij Duurstede, The Netherlands). At the start of the 12-week intervention period, mice were stratified based on body weight over 7 groups ( n = 12), to obtain ident..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Motor performance",
"text": "Several parameters of gait were assessed in week 10 by CatWalk analysis (Noldus Information Technology, Wageningen, The Netherlands) using the reflection of light projected on a glass walking area. Each mouse made at least six compliant runs, being defined as a maximum speed variation of 40 %, minimum run duration of 0.5 s and maximum run duration of 10 s. Quantitative gait parameters were analysed using the CatWalk XT 10.0 software (Noldus Information Technology)."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Lipid determination in serum and liver",
"text": "Because flavonoids were previously shown to interfere with commonly used commercially available enzymatic lipid assays (Hoek-van den Hil et al. ), alternative methods were used to measure the amount of lipids in serum and liver, as described (Hoek-van den Hil et al. ). Serum lipids were extracted and analysed with 1 H nuclear magnetic resonance ( 1 H-NMR), and neutral lipids were stained in frozen liver sections with Oil red O (Sigma) and quantified."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Statistical analysis",
"text": "All statistical analyses were in principle done based on all 12 mice per group with the following exceptions: HF + Q only for 11 mice; for indirect calorimetry measurements ( n = 9), histological stainings ( n = 6), faeces collection ( n = 4) and serum flavonoid measurements ( n = 6), subsets of mice as indicated between the brackets were randomly selected, because of limited equipment or practical reasons. GraphPad Prism version 5.03 (GraphPad software, San Diego, CA, USA) was used for statistical analysis. Data were checked for normality and if needed log transformed ( Acot3, Cyp4a14, Por, Fasn and Cyp2b9 in liver and Cpt1a in EpiWAT). One-way ANOVA was used to compare the groups, followed by Dunnett's post hoc test to compare the different HF + flavonoid groups to HF, and HF to NF. For the analysis of data of..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice",
"datePublished": "2015",
"author": [
{
"@type": "Person",
"name": "Elise F. Hoek-van den Hil"
},
{
"@type": "Person",
"name": "Evert M. van Schothorst"
},
{
"@type": "Person",
"name": "Inge van der Stelt"
},
{
"@type": "Person",
"name": "Hans J. M. Swarts"
},
{
"@type": "Person",
"name": "Marjanne van Vliet"
},
{
"@type": "Person",
"name": "Tom Amolo"
},
{
"@type": "Person",
"name": "Jacques J. M. Vervoort"
},
{
"@type": "Person",
"name": "Dini Venema"
},
{
"@type": "Person",
"name": "Peter C. H. Hollman"
},
{
"@type": "Person",
"name": "Ivonne M. C. M. Rietjens"
},
{
"@type": "Person",
"name": "Jaap Keijer"
}
],
"identifier": "10.1007/s12263-015-0469-z"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Direct comparison of metabolic health effects of the flavonoids quercetin, hesperetin, epicatechin, apigenin and anthocyanins in high-fat-diet-fed mice methods",
"item": "https://replicatescience.com/experiments/direct-comparison-of-metabolic-health-effects-of-the-flavonoids-quercetin-hesperetin-epicatechin-apigenin-and-anthocyanins-in-high-fat-diet-fed-mice-methods-elise-f-hoek-van-den-hi/direct-comparison-of-metabolic-health-effects-of-the-flavonoids-quercetin-hesperetin-epicatechin-api-mlph92i2"
}
]
}
]