Oral Supplementation With a Bovine Thymus Extract Reduces Neuronal Excitability in Aging 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?
Materials and Methods
A total of 45 mice were used in this study. Adult male C57BL/6 mice (12 months of age) were purchased from Charles River Laboratories (RRID:MGI:7264769; Wilmington, MA, USA). Because the first evidence for TNF's potential to modulate CNS function came from studies conducted in males and needed further validation, female mice were not included in the current study [ ]. Animals were received at the vivarium of the College of Staten Island/City University of New York (CUNY) and assigned unique identification numbers. Animals were housed individually in ventilated cages and were acclimated for 7 days prior to initiation of the study. All mice were examined, handled, and weighed prior to the study initiation to assure adequate health and suitability. During the study, 12/12 h light/dark cycles were maintained. The room temperature was maintained between 20°C and 23&#...
Thymus Nuclear Fraction (TNF) Supplement
The powder was homogeneously dispersed in sterile water and administered by oral gavage twice a day at a dose volume of 3.5 mL/kg. Fifteen mice were used in each treatment group. Group 1: treatment at low dose of 170 mg/kg twice a day (340 mg/kg/day); Group 2: treatment at high dose of 340 mg/kg twice a day (680 mg/kg/day); Group 3: vehicle control (water administered at a dose volume of 3.5 mg/kg).
Western Blot
At the end of the study, mice were sacrificed by decapitation and brains were immediately dissected within 1 min, frozen on dry ice and stored at -80°C. Total soluble and membrane bound proteins were extracted [ ] and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, Rockford, IL, USA). For GABA receptor β2 and β3 subunits detection, 15 µg of protein was loaded onto a Mini-PROTEAN TGX 4%-16% gradient polyacrylamide-SDS gel (Bio-Rad Laboratories Inc) and transferred onto PVDF membrane (Millipore, Bedford, MA) after electrophoresis. Membranes were sequentially probed with two antibodies: a monoclonal antibody that recognizes β2 and β3 subunits of GABA A receptor (Clone 62-3G1; Millipore, Temecula, CA) and a rabbit polyclonal antibody (A2668, Sigma, St. Louis, MO) that...
Open Field Test
The open field was constructed of a 62 cm by 62 cm laminated wood floor surrounded by clear acrylic walls of 30 cm height. A 4 × 4 grid matrix was painted on the floor of the open field, dividing the field into 16 squares (12 outer, 4 inner) each measuring 15.5 cm by 15.5 cm. The experimenter was blinded to mouse treatment. The light in the behavior testing room was maintained at 200 lx. At the beginning of the open field session, mice were placed on the right front corner square (relative to the position of the experimenter) and were retained inside a clear acrylic tube (diameter = 3.5 in.). After 30 s, the tube was lifted and the mouse was allowed to move freely about the open field for a total period of 5 min, then returned to its home cage. The open field was washed with Windex between each session....
Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose
The observed increases in the expression of GABAergic system proteins in aging TNF-supplemented animals may suggest possible alterations in mouse behaviors related to alertness or locomotion [,, ]. We therefore examined the activity of control and TNF-supplemented mice using an open field behavioral test. We found that the time spent immobile (the inverse of the time spent moving) did not significantly differ between the treatment groups at baseline or at week 8 of the study (Figure ). Two-Way Repeated Measures ANOVA showed significant treatment × time interaction for total distance traveled (F[2,34] = 4.195, p = 0.0235, partial eta squared = 0.198) and average travel speed (F[2,34] = 4.185, p = 0.0237, partial eta squared = 0.192). Total distance traveled and the avera...
Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose
Locomotor activity on an open field test in control (CON) mice and mice supplemented with TNF at a low dose (LD) or a high dose (HD). (A) No significant changes were detected between the three groups in times spent immobile at baseline or at 8 weeks of supplementation. (B) Total distance traveled during a 5-min test was not different between the groups at baseline but remained increased in HD group animals compared to CON and LD groups ( p < 0.05) at week 8 of supplementation. (C) The average travel speed did not differ between the groups at baseline, but was significantly higher in HD compared to CON and LD group animals ( p < 0.05). *-Significantly different from CON and LD, p < 0.05; ns-not significant by Two-Way Repeated Measures ANOVA and Tukey's post hoc test.
Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose
Changes in body weights of control (CON) mice and mice supplemented with TNF at a low dose (LD) or high dose (HD). Animals in the HD group displayed a trend towards reduction in average body weight during the supplementation period compared to both CON and LD mice.
Thymus Extract Supplementation Reduces Neuronal Excitability
Representative recordings of field potentials in the prefrontal cortex. Spontaneous electrophysiological activity was measured in the prefrontal cortices of control mice (CON) and mice supplemented with TNF at a low-dose (LD) or a high-dose (HD) (A, C and E, upper traces). Field potentials were recorded for 15 min, and the resulting traces were filtered between 0.4 and 500 Hz and amplified. (A, C, E) The original traces (green-upper) and filtered traces (black-slow ripples 125-250 Hz; red-fast ripples 250-500 Hz). (B, D, F) The power densities of field potentials and filtered frequencies were converted into heat maps for control animals (B) and animals supplemented at a low dose (D) or a high dose (F).
Measurement outputs
What raw and processed outputs should exist?
Protein expression by western blot
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
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: Protein expression by western blot.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Protein expression by western blot.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (8)
A total of 45 mice were used in this study. Adult male C57BL/6 mice (12 months of age) were purchased from Charles River Laboratories (RRID:MGI:7264769; Wilmington, MA, USA). Because the first evidence for TNF's potential to modulate CNS function came from studies conducted in males and needed further validation, female mice were not included in the current study [ ]. Animals were received at the vivarium of the College of Staten Island/City University of New York (CUNY) and assigned unique identification numbers. Animals were housed individually in ventilated cages and were acclimated for 7 days prior to initiation of the study. All mice were examined, handled, and weighed prior to the study initiation to assure adequate health and suitability. During the study, 12/12 h light/dark cycles were maintained. The room temperature was maintained between 20°C and 23°C with a relative humidity around 50%. Chow (Laboratory Rodent Diet 5001; Cat.#0001319; LabDiet, St. Louis, MO, USA) and water were provided ad libitum for the duration of the study. Animals were randomly assigned across treatment groups. Body weights were taken twice a week during the stud...
The powder was homogeneously dispersed in sterile water and administered by oral gavage twice a day at a dose volume of 3.5 mL/kg. Fifteen mice were used in each treatment group. Group 1: treatment at low dose of 170 mg/kg twice a day (340 mg/kg/day); Group 2: treatment at high dose of 340 mg/kg twice a day (680 mg/kg/day); Group 3: vehicle control (water administered at a dose volume of 3.5 mg/kg).
At the end of the study, mice were sacrificed by decapitation and brains were immediately dissected within 1 min, frozen on dry ice and stored at -80°C. Total soluble and membrane bound proteins were extracted [ ] and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, Rockford, IL, USA). For GABA receptor β2 and β3 subunits detection, 15 µg of protein was loaded onto a Mini-PROTEAN TGX 4%-16% gradient polyacrylamide-SDS gel (Bio-Rad Laboratories Inc) and transferred onto PVDF membrane (Millipore, Bedford, MA) after electrophoresis. Membranes were sequentially probed with two antibodies: a monoclonal antibody that recognizes β2 and β3 subunits of GABA A receptor (Clone 62-3G1; Millipore, Temecula, CA) and a rabbit polyclonal antibody (A2668, Sigma, St. Louis, MO) that recognizes β-actin (1:10,000 dilution) as a control for protein loading. For GAD detection, 10 µg of protein was used, and membranes were simultaneously probed with two antibodies: SC-365180, a monoclonal (1:5000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) that re...
The open field was constructed of a 62 cm by 62 cm laminated wood floor surrounded by clear acrylic walls of 30 cm height. A 4 × 4 grid matrix was painted on the floor of the open field, dividing the field into 16 squares (12 outer, 4 inner) each measuring 15.5 cm by 15.5 cm. The experimenter was blinded to mouse treatment. The light in the behavior testing room was maintained at 200 lx. At the beginning of the open field session, mice were placed on the right front corner square (relative to the position of the experimenter) and were retained inside a clear acrylic tube (diameter = 3.5 in.). After 30 s, the tube was lifted and the mouse was allowed to move freely about the open field for a total period of 5 min, then returned to its home cage. The open field was washed with Windex between each session. The behavioral parameter measured was the total number of grid crossings made during the 5-min trial. A crossing was defined as all four paws moving out of one of the 16 squares and into another. This measure is expected to correlate highly with the distance traveled in the open field, and it...
The observed increases in the expression of GABAergic system proteins in aging TNF-supplemented animals may suggest possible alterations in mouse behaviors related to alertness or locomotion [,, ]. We therefore examined the activity of control and TNF-supplemented mice using an open field behavioral test. We found that the time spent immobile (the inverse of the time spent moving) did not significantly differ between the treatment groups at baseline or at week 8 of the study (Figure ). Two-Way Repeated Measures ANOVA showed significant treatment × time interaction for total distance traveled (F[2,34] = 4.195, p = 0.0235, partial eta squared = 0.198) and average travel speed (F[2,34] = 4.185, p = 0.0237, partial eta squared = 0.192). Total distance traveled and the average travel speed did not differ between all three groups at baseline (Figure ). However, mice supplemented with TNF at a high dose (HD group) traveled significantly longer distances and maintained significantly higher travel speed at week 8 of supplementation compared to LD and control group an...
Locomotor activity on an open field test in control (CON) mice and mice supplemented with TNF at a low dose (LD) or a high dose (HD). (A) No significant changes were detected between the three groups in times spent immobile at baseline or at 8 weeks of supplementation. (B) Total distance traveled during a 5-min test was not different between the groups at baseline but remained increased in HD group animals compared to CON and LD groups ( p < 0.05) at week 8 of supplementation. (C) The average travel speed did not differ between the groups at baseline, but was significantly higher in HD compared to CON and LD group animals ( p < 0.05). *-Significantly different from CON and LD, p < 0.05; ns-not significant by Two-Way Repeated Measures ANOVA and Tukey's post hoc test.
Changes in body weights of control (CON) mice and mice supplemented with TNF at a low dose (LD) or high dose (HD). Animals in the HD group displayed a trend towards reduction in average body weight during the supplementation period compared to both CON and LD mice.
Representative recordings of field potentials in the prefrontal cortex. Spontaneous electrophysiological activity was measured in the prefrontal cortices of control mice (CON) and mice supplemented with TNF at a low-dose (LD) or a high-dose (HD) (A, C and E, upper traces). Field potentials were recorded for 15 min, and the resulting traces were filtered between 0.4 and 500 Hz and amplified. (A, C, E) The original traces (green-upper) and filtered traces (black-slow ripples 125-250 Hz; red-fast ripples 250-500 Hz). (B, D, F) The power densities of field potentials and filtered frequencies were converted into heat maps for control animals (B) and animals supplemented at a low dose (D) or a high dose (F).
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Oral Supplementation With a Bovine Thymus Extract Reduces Neuronal Excitability in Aging Mice methods",
"description": "Protocol for Oral Supplementation With a Bovine Thymus Extract Reduces Neuronal Excitability in Aging Mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Materials and Methods",
"text": "A total of 45 mice were used in this study. Adult male C57BL/6 mice (12 months of age) were purchased from Charles River Laboratories (RRID:MGI:7264769; Wilmington, MA, USA). Because the first evidence for TNF's potential to modulate CNS function came from studies conducted in males and needed further validation, female mice were not included in the current study [ ]. Animals were received at the vivarium of the College of Staten Island/City University of New York (CUNY) and assigned unique identification numbers. Animals were housed individually in ventilated cages and were acclimated for 7 days prior to initiation of the study. All mice were examined, handled, and weighed prior to the study initiation to assure adequate health and suitability. During the study, 12/12 h light/dark cycles were maintained. The room temperature was maintained between 20°C and 23&#..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Thymus Nuclear Fraction (TNF) Supplement",
"text": "The powder was homogeneously dispersed in sterile water and administered by oral gavage twice a day at a dose volume of 3.5 mL/kg. Fifteen mice were used in each treatment group. Group 1: treatment at low dose of 170 mg/kg twice a day (340 mg/kg/day); Group 2: treatment at high dose of 340 mg/kg twice a day (680 mg/kg/day); Group 3: vehicle control (water administered at a dose volume of 3.5 mg/kg)."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Western Blot",
"text": "At the end of the study, mice were sacrificed by decapitation and brains were immediately dissected within 1 min, frozen on dry ice and stored at -80°C. Total soluble and membrane bound proteins were extracted [ ] and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, Rockford, IL, USA). For GABA receptor β2 and β3 subunits detection, 15 µg of protein was loaded onto a Mini-PROTEAN TGX 4%-16% gradient polyacrylamide-SDS gel (Bio-Rad Laboratories Inc) and transferred onto PVDF membrane (Millipore, Bedford, MA) after electrophoresis. Membranes were sequentially probed with two antibodies: a monoclonal antibody that recognizes β2 and β3 subunits of GABA A receptor (Clone 62-3G1; Millipore, Temecula, CA) and a rabbit polyclonal antibody (A2668, Sigma, St. Louis, MO) that..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Open Field Test",
"text": "The open field was constructed of a 62 cm by 62 cm laminated wood floor surrounded by clear acrylic walls of 30 cm height. A 4 × 4 grid matrix was painted on the floor of the open field, dividing the field into 16 squares (12 outer, 4 inner) each measuring 15.5 cm by 15.5 cm. The experimenter was blinded to mouse treatment. The light in the behavior testing room was maintained at 200 lx. At the beginning of the open field session, mice were placed on the right front corner square (relative to the position of the experimenter) and were retained inside a clear acrylic tube (diameter = 3.5 in.). After 30 s, the tube was lifted and the mouse was allowed to move freely about the open field for a total period of 5 min, then returned to its home cage. The open field was washed with Windex between each session...."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose",
"text": "The observed increases in the expression of GABAergic system proteins in aging TNF-supplemented animals may suggest possible alterations in mouse behaviors related to alertness or locomotion [,, ]. We therefore examined the activity of control and TNF-supplemented mice using an open field behavioral test. We found that the time spent immobile (the inverse of the time spent moving) did not significantly differ between the treatment groups at baseline or at week 8 of the study (Figure ). Two-Way Repeated Measures ANOVA showed significant treatment × time interaction for total distance traveled (F[2,34] = 4.195, p = 0.0235, partial eta squared = 0.198) and average travel speed (F[2,34] = 4.185, p = 0.0237, partial eta squared = 0.192). Total distance traveled and the avera..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose",
"text": "Locomotor activity on an open field test in control (CON) mice and mice supplemented with TNF at a low dose (LD) or a high dose (HD). (A) No significant changes were detected between the three groups in times spent immobile at baseline or at 8 weeks of supplementation. (B) Total distance traveled during a 5-min test was not different between the groups at baseline but remained increased in HD group animals compared to CON and LD groups ( p < 0.05) at week 8 of supplementation. (C) The average travel speed did not differ between the groups at baseline, but was significantly higher in HD compared to CON and LD group animals ( p < 0.05). *-Significantly different from CON and LD, p < 0.05; ns-not significant by Two-Way Repeated Measures ANOVA and Tukey's post hoc test."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Locomotor Activity Is Preserved in Aging Mice Supplemented With TNF at High Dose",
"text": "Changes in body weights of control (CON) mice and mice supplemented with TNF at a low dose (LD) or high dose (HD). Animals in the HD group displayed a trend towards reduction in average body weight during the supplementation period compared to both CON and LD mice."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Thymus Extract Supplementation Reduces Neuronal Excitability",
"text": "Representative recordings of field potentials in the prefrontal cortex. Spontaneous electrophysiological activity was measured in the prefrontal cortices of control mice (CON) and mice supplemented with TNF at a low-dose (LD) or a high-dose (HD) (A, C and E, upper traces). Field potentials were recorded for 15 min, and the resulting traces were filtered between 0.4 and 500 Hz and amplified. (A, C, E) The original traces (green-upper) and filtered traces (black-slow ripples 125-250 Hz; red-fast ripples 250-500 Hz). (B, D, F) The power densities of field potentials and filtered frequencies were converted into heat maps for control animals (B) and animals supplemented at a low dose (D) or a high dose (F)."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Oral Supplementation With a Bovine Thymus Extract Reduces Neuronal Excitability in Aging Mice",
"datePublished": "2026",
"author": [
{
"@type": "Person",
"name": "Abdeslem El-Idrissi"
},
{
"@type": "Person",
"name": "Natalia Surzenko"
},
{
"@type": "Person",
"name": "Bassem F. El-Khodor"
}
],
"identifier": "10.1096/fba.2025-00256"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Oral Supplementation With a Bovine Thymus Extract Reduces Neuronal Excitability in Aging Mice methods",
"item": "https://replicatescience.com/experiments/oral-supplementation-with-a-bovine-thymus-extract-reduces-neuronal-excitability-in-aging-mice-methods-abdeslem-el-8208-idrissi-pmc12857519/oral-supplementation-with-a-bovine-thymus-extract-reduces-neuronal-excitability-"
}
]
}
]