Materials & Equipment Checklist
10 items1 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment1
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Protocol Steps
1
Animal housing and acclimation
Male BKS.Cg-Dock7m+/+Leprdb/J mice obtained from The Jackson Laboratory and housed in animal facility under standard conditions
22 ± 2°C
Note: Strict 12h:12h light:dark cycle maintained; humidity at 50 ± 15%
View evidence from paper
“All mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the Northwest A&F University animal facility under standard conditions with a strict 12 h:12 h light:dark cycle, humidity at 50 ± 15%, temperature 22 ± 2°C”
2
Baseline feeding and monitoring
Mice fed ad libitum with regular chow (AIN-93M) and pure water before intervention at 4 months of age
Note: Bodyweight, food intake, and water consumption recorded on fasting day
View evidence from paper
“The animals were fed ad libitum before the IF was initiated at 4 months of age. Mice were fed a regular chow (AIN-93M, purchased from TROPHIC Animal Feed High-tech Co. Ltd, Nantong, China) and pure water. Bodyweight, food intake, and water consumption were recorded on fasting day”
3
Intermittent fasting regimen initiation
Mice divided into groups and subjected to intermittent fasting protocol: food deprivation for 24 hours every other day with ad libitum feeding on intervening days
28 days
Note: Applied to db/m and db/db mice in multiple cohorts
View evidence from paper
“the IF mice were deprived of food for 24 h, every other day and were fed ad libitum on the intervening day for 28 days”
4
Antibiotics treatment initiation
Antibiotics cocktail administered in drinking water to deplete gut microbiota in designated groups
Starting 14 days before IF regimen and throughout experiment
Note: Different concentrations used for db/db mice (full strength) and db/m mice (half concentration)
View evidence from paper
“The antibiotics cocktail...were given in the drinking water starting 14 days before the IF regimen and throughout the experiment”
5
Metabolite treatment administration
For Set 4 animals, IPA, 5-HT, and TUDCA administered via intraperitoneal injection; SCFAs dissolved in drinking water
14 days treatment during behavioral tests
Note: Control groups received saline injections of same volume
View evidence from paper
“For db/db-IPA, db/db-5-HT, db/db-TUDCA groups, mice were intraperitoneally injected with IPA (10 mg kg−1 d−1), 5-HT (1 mg kg−1 d−1), and TUDCA (250 mg kg−1 d−1) dissolved in saline, respectively. The mice in other groups were injected with same volume saline”
6
Microbiota depletion verification
16S rRNA copies in feces detected via qPCR to confirm antibiotics effectiveness
View evidence from paper
“The 16S rRNA copies in the feces of the animals after antibiotics treatment were detected with qPCR”
7
Water maze behavioral testing
Cognitive behavioral assessment performed to evaluate spatial learning and memory
Note: All behavioral studies conducted on fasting day of IF regimen. One mouse in db/db group drowned during test and data excluded
View evidence from paper
“After IF regimen, the antibiotics treatment, and metabolites treatment, the cognitive behavioral assessment was determined with a water-maze test. All of the behavioral studies and biochemical samples were collected/detected on the fasting day of IF regimen”
8
Sample collection
After behavioral testing, mice euthanized and serum and tissue samples collected
Note: Samples either snap-frozen in liquid nitrogen and stored at -80°C or stored in 4% paraformaldehyde for histological analysis
View evidence from paper
“After behavioral tests, mice were killed, then the serum and tissues samples were collected by either snap-frozen by liquid nitrogen and store at −80°C or directly stored in 4% paraformaldehyde for histological analysis”
9
Multi-OMICS analysis (Set 2 only)
Fecal, serum, and hippocampus samples analyzed for gut microbiome, metabolome, and RNA sequencing
View evidence from paper
“Fecal, serum, and hippocampus samples were collected to analyze gut microbiome, metabolome, and RNA sequencing, respectively”
Subjects / Specimens
- Species
- mouse
- Strain
- BKS.Cg-Dock7m+/+Leprdb/J and heterozygous controls
- Age
- 4 months at start of intervention
- Sex
- male
Homozygous Leprdb/db mice were diabetic; heterozygous Leprdb/m mice were used as controls. Mice obtained from The Jackson Laboratory (Bar Harbor, ME)