Objective: To evaluate the effects of lifelong caloric restriction (60% of ad libitum intake) on longevity, body weight, and food intake in C57BL/6 and B6C3F1 mice, with comparison to standard diet and metformin-supplemented diet groups
This is a Caloric Restriction Study protocol using mouse as the model organism. The procedure involves 14 procedural steps, 3 equipment items, 6 materials. Extracted from a 2013 paper published in Nature Communications.
Model and subjects
mouse • C57BL/6 and B6C3F1 • male • 6 months (0.1% metformin and CR groups) or 11 months (1% metformin dose) at study start; treatment began at 27 weeks of age for CR • [object Object]
Study window
~27 week study window
Core workflow
Animal acquisition and initial housing • Pre-treatment diet standardization for C57BL/6 mice • Treatment group assignment for B6C3F1 mice
Primary readouts
Key equipment and reagents
Verified items
0
Direct vendor links
0
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Male C57BL/6 mice were purchased from Charles Rivers (National Institute on Aging Aged Rodent Colony) at either 6 months of age (for 0.1% metformin and CR groups) or 11 months of age (for 1% metformin dose). B6C3F1 male mice were purchased from Harlan Breeders (Indianapolis).
Note: Mice were maintained on light:dark 12:12-h schedule
“Male C57BL/6 mice at either 6 months of age (for 0.1% metformin and CR) or 11 months of age (for 1% metformin dose) were purchased from the National Institute on Aging Aged Rodent Colony from Charles Rivers”
C57BL/6 mice were maintained on standard purified AIN-93G diet from acquisition until they reached one year of age, when treatments were to begin.
Note: This standardization period ensured all mice had equivalent baseline nutritional status before treatment assignment
“The C57BL/6 mice were maintained on a standard purified mouse diet (AIN-93G) until they reached one year of age when the treatments started”
B6C3F1 male mice were randomly assigned to treatment groups at 12 months of age. Mice were shifted from ad libitum chow feeding (Purina Mills Diet #5001) to daily feeding with measured quantities of control diet.
Note: Random assignment ensured unbiased group distribution
“male mice (Harlan Breeders; Indianapolis) were randomly assigned to treatment groups at 12 months of age”
C57BL/6 mice in the CR group were subjected to lifelong restriction on AIN-93G diet, starting at 27 weeks of age, with a daily food allotment of 60% of that eaten by ad libitum animals.
Note: CR was maintained for the remainder of the animals' lives
“The CR animals were subjected to a lifelong restriction on AIN-93G diet, starting at 27 weeks of age, with a daily food allotment of 60% of that eaten by the ad lib animals”
C57BL/6 mice in the SD (standard diet) group were fed AIN-93G diet ad libitum for the remainder of their lives.
Note: Ad libitum feeding served as control for CR group
“the C57BL/6 groups in this study were fed the standard AIN-93G diet (SD)”
C57BL/6 mice were fed AIN-93G diet supplemented with either 0.1% or 1% metformin ad libitum for the remainder of their lives. Pure metformin was obtained from Farmhispania S.A. (Barcelona, Spain) and mixed to homogeneity during diet manufacturing by Dyets Inc. (Bethlehem, PA).
Note: Two metformin dose groups: 0.1% and 1% w/w in diet
“the C57BL/6 groups in this study were fed the standard AIN-93G diet (SD) or AIN-93G plus 0.1% metformin or 1% metformin ad lib for the remainder of their lives”
B6C3F1 control group mice were shifted to daily feeding with 13.3 kcal/day/mouse of AIN-93M control diet (Diet No. F05312, BioServ, Frenchtown, NJ).
Note: Precise daily caloric amount was measured and provided
“In the control group, mice were shifted from ad lib chow feeding (Diet # 5001, Purina Mills, Richmond, IN) to daily feeding with 13.3 kcal/day/mouse of control diet (AIN-93M, Diet No. F05312; BioServ, Frenchtown, NJ)”
B6C3F1 metformin group mice were shifted to daily feeding with an identical quantity (13.3 kcal/day/mouse) of AIN-93M diet supplemented with metformin at 100 mg/kg diet (0.1% w/w), approximately 10.6 mg/kg body weight/day. Metformin was obtained from Caraco Pharmaceutical Laboratories (Detroit, MI), mixed with powdered diet, and cold-pressed into 1-gram pellets by BioServ.
Note: Metformin dose was 0.1% w/w in diet
“For the metformin B6C3F1 group, mice were shifted to daily feeding with an identical quantity of control AIN-93M diet supplemented with metformin (Caraco Pharmaceutical Laboratories, Detroit, MI) at 100 mg/kg diet (0.1% w/w)”
Metformin was mixed with powdered diet and cold-pressed into 1-gram pellets by BioServ. Chow was produced every 3 months during the length of the study, never permitted to exceed 50°C, and kept away from light whenever possible to ensure metformin stability. Stock metformin and all chow were stored in the dark at 4°C.
Note: Light/dark cycle in mouse facility was not altered to protect metformin stability
“The chow was produced every 3 months during the length of the study, never was permitted to exceed 50 °C and was kept away from light whenever possible to ensure the stability of metformin”
All mice were fed daily. With rare exceptions, all food was eaten each day.
Note: Complete food consumption was nearly universal
“All mice were fed daily and, with rare exceptions, all food was eaten each day”
Food intake and body weight were measured on a biweekly or bimonthly basis for the duration of the study.
Note: Regular monitoring of these parameters tracked treatment effects
“Food intake and body weight were measured on a biweekly or bimonthly basis for the duration of the study”
Criteria for euthanasia were based on independent assessment by a veterinarian according to AAALAC guidelines. Only cases where the condition of the animal was considered incompatible with continued survival were euthanized and represented in survival curves.
Note: Veterinary assessment ensured humane treatment and consistent criteria
“The criteria for euthanasia was based on an independent assessment by a veterinarian, according to AAALAC guidelines and only cases, where the condition of the animal was considered incompatible with continued survival, are represented in the curves”
Every animal found dead or euthanized was necropsied for pathology score.
Note: Pathology scoring provided additional outcome data
“Every animal found dead or euthanized was necropsied for pathology score”
Survival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Note: Kaplan-Meier method accounts for censored data and varying follow-up times
“Survival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To evaluate the effects of lifelong caloric restriction (60% of ad libitum intake) on longevity, body weight, and food intake in C57BL/6 and B6C3F1 mice, with comparison to standard diet and metformin-supplemented diet groups
Objective
To evaluate the effects of lifelong caloric restriction (60% of ad libitum intake) on longevity, body weight, and food intake in C57BL/6 and B6C3F1 mice, with comparison to standard diet and metformin-supplemented diet groups
Subjects
From papermouse • C57BL/6 and B6C3F1 • male • 6 months (0.1% metformin and CR groups) or 11 months (1% metformin dose) at study start; treatment began at 27 weeks of age for CR
Sample count
From paper[object Object]
Cohort notes
From paperMale C57BL/6 mice purchased at either 6 months or 11 months of age; B6C3F1 mice randomly assigned to treatment groups at 12 months of age
Animal acquisition and initial housing (Not specified)
Pre-treatment diet standardization for C57BL/6 mice (Until mice reached one year of age)
Treatment group assignment for B6C3F1 mice (At 12 months of age)
Initiate caloric restriction treatment for C57BL/6 CR group (Starting at 27 weeks of age, lifelong)
Survival curves and longevity
From paperSurvival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Body weight (measured biweekly or bimonthly)
From paperSurvival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Food intake (measured biweekly or bimonthly)
From paperSurvival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Pathology scores from necropsy
From paperSurvival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Survival curves and longevity
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Body weight (measured biweekly or bimonthly)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Food intake (measured biweekly or bimonthly)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Pathology scores from necropsy
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Survival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Scoring or quantification
Quantify the primary readouts for this experiment: Survival curves and longevity; Body weight (measured biweekly or bimonthly); Food intake (measured biweekly or bimonthly); Pathology scores from necropsy.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Survival curves and longevity, Body weight (measured biweekly or bimonthly), Food intake (measured biweekly or bimonthly), Pathology scores from necropsy.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Alejandro Martin-Montalvo et al. (2013). Metformin improves healthspan and lifespan in mice. Nature Communications
Animal acquisition and initial housing • Protocol step
“Male C57BL/6 mice at either 6 months of age (for 0.1% metformin and CR) or 11 months of age (for 1% metformin dose) were purchased from the National Institute on Aging Aged Rodent Colony from Charles Rivers”
Pre-treatment diet standardization for C57BL/6 mice • Protocol step
“The C57BL/6 mice were maintained on a standard purified mouse diet (AIN-93G) until they reached one year of age when the treatments started”
Treatment group assignment for B6C3F1 mice • Protocol step
“male mice (Harlan Breeders; Indianapolis) were randomly assigned to treatment groups at 12 months of age”
Initiate caloric restriction treatment for C57BL/6 CR group • Protocol step
“The CR animals were subjected to a lifelong restriction on AIN-93G diet, starting at 27 weeks of age, with a daily food allotment of 60% of that eaten by the ad lib animals”
Direct vendor pages are linked from the protocol above. This section stays focused on the full comparison view and the prep checklist.
Gather these items before starting the experiment. Check off items as you prepare.
Not specified • Not specified • Not specified • Not specified
Not specified • Not specified • Not specified • Not specified
Not specified • Not specified • Not specified • Not specified
Not specified • Not specified • Not specified • Not specified
BioServ • Not specified • Diet No. F05312 • Not specified
Purina Mills • Not specified • Diet # 5001 • Not specified
Farmhispania S.A. (for C57BL/6 groups); Caraco Pharmaceutical Laboratories (for B6C3F1 groups) • Not specified • Not specified • Not specified
BioServ • Not specified • Not specified • Not specified
Dyets Inc. • Not specified • Not specified • Not specified
Statistical method (not software) • Not specified
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