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MB Sample ID: SA210151
Local Sample ID: | Terence_MS14_Quad_Aq |
Subject ID: | SU002275 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 13 weeks |
Weight Or Weight Range: | (28.4±1.1 g (control SHAM mice) vs 26.4±2.1 g (control AVF mice) vs 23.6±2.1 (CKD SHAM mice) vs 24.7±1.3 (CKD AVF mice), N=8-10/group). |
Gender: | Male |
Animal Animal Supplier: | Jackson Labs (Stock # 000664) |
Animal Housing: | Housed in a temperature of 22 oC |
Animal Light Cycle: | 12-hour light/12-hour dark |
Animal Feed: | Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) |
Animal Water: | free access to food and water (3-5 animals per cage). |
Animal Inclusion Criteria: | (3-5 animals per cage). |
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Subject:
Subject ID: | SU002275 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Genotype Strain: | C57BL/6J |
Age Or Age Range: | 13 weeks |
Weight Or Weight Range: | (28.4±1.1 g (control SHAM mice) vs 26.4±2.1 g (control AVF mice) vs 23.6±2.1 (CKD SHAM mice) vs 24.7±1.3 (CKD AVF mice), N=8-10/group). |
Gender: | Male |
Animal Animal Supplier: | Jackson Labs (Stock # 000664) |
Animal Housing: | Housed in a temperature of 22 oC |
Animal Light Cycle: | 12-hour light/12-hour dark |
Animal Feed: | Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) |
Animal Water: | free access to food and water (3-5 animals per cage). |
Animal Inclusion Criteria: | (3-5 animals per cage). |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Terence_MS14_Quad_Aq | SA210151 | FL025645 | CON_AVF | Factor1*** |
Collection:
Collection ID: | CO002268 |
Collection Summary: | Two weeks after surgery, mice were anesthetized using isoflurane and the left quadriceps muscle was rapidly dissected and immediately frozen in liquid nitrogen and stored at -80oC for metabolite extraction. Euthanasia was carried out by thoracotomy followed by cervical dislocation. |
Sample Type: | Muscle |
Collection Method: | While under isoflurane anesthesia, tissues were rapidly dissected and snap frozen in liquid nitrogen and stored at -80°C until metabolite extraction |
Collection Location: | University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, Gainesville, FL 32611 |
Storage Conditions: | -80℃ |
Storage Vials: | cryovials |
Treatment:
Treatment ID: | TR002287 |
Treatment Summary: | Briefly, mice were acclimated to a standard casein-based chow (Control Diet) for 7 days. Thereafter, the animals were randomly assign to either remain on Control Diet (non-CKD groups) or receive adenine-supplemented diet to induce CKD. Following three weeks of the casein/adenine diet, animals were randomly assigned to receive either an arteriovenous fistula surgery (AVF) or sham surgery (SHAM) |
Animal Vet Treatments: | none |
Animal Anesthesia: | isoflurane |
Animal Fasting: | non-fasted |
Animal Endp Euthanasia: | Euthanasia was carried out by thoracotomy followed by cervical dislocation. |
Animal Endp Tissue Coll List: | Skeletal muscle (quadriceps) |
Sample Preparation:
Sampleprep ID: | SP002281 |
Sampleprep Summary: | Weights of frozen quadriceps specimens were weighed using a microbalance (Mettler-Toledo, Columbus, OH, USA). Next, a slightly modified FOLCH extraction was performed to extract aqueous and lipid phase metabolites. The aqueous phase was lyophilized overnight (Labconco Corporation, Kansas, MO, USA) and the lipid phase was dried by passing inert nitrogen gas. The resulting aqueous and lipid phase dry powders were stored at -80oC until analysis using nuclear magnetic resonance (NMR). The dry powder of aqueous phase samples was dissolved in 50 µL of phosphate buffer system (50 mM, pH 7.2) consisting of 0.5 mM D6-DSS, 2 mM EDTA and 0.2% NaN2. Lipid phase dry powders were dissolved in 70 µL of CDCl3 supplemented with 10 mM of pyrazine (as internal NMR standard). All samples were loaded into 1.5 mm optical density (O.D.) NMR tubes. |
Sampleprep Protocol Filename: | AVF_NMR_Aqueous_Phase_Procedures.docx |
Processing Method: | Lyophilization and Homogenization |
Processing Storage Conditions: | -80℃ |
Extraction Method: | Modified FOLCH extraction |
Extract Storage: | -80℃ |
Sample Resuspension: | In 50 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS and 0.2% sodium azide for aqueous phase samples. |
Sample Spiking: | 0.5 mM of DSS for aqueous phase samples |
Analysis:
MB Sample ID: | SA210151 |
Analysis ID: | AN003583 |
Laboratory Name: | Terence lab, UF |
Analysis Type: | NMR |
Acquisition Date: | 09/14/2021 |
Software Version: | Bruker Topspin |
Operator Name: | Ram Khattri |
Detector Type: | Bruker |
Data Format: | fid, 1r |
Num Factors: | 5 |
Num Metabolites: | 37 |
Units: | mM |
NMR:
NMR ID: | NM000241 |
Analysis ID: | AN003583 |
Instrument Name: | Bruker Avance III Cryo800 MHz 54 mm |
Instrument Type: | FT-NMR |
NMR Experiment Type: | 1D-1H |
Field Frequency Lock: | Deuterium |
Standard Concentration: | 0.5 mM DSS |
Spectrometer Frequency: | 799.9 |
NMR Probe: | CP TXI CryoProbe |
NMR Solvent: | Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in deuterated environment |
NMR Tube Size: | 1.5 mm O.D. |
Shimming Method: | Topshim |
Pulse Sequence: | noesypr1d |
Water Suppression: | presat |
Pulse Width: | 90-degree |
Receiver Gain: | 645 |
Offset Frequency: | 2827.31 Hz |
Chemical Shift Ref Cpd: | DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) |
Temperature: | 25 o C |
Number Of Scans: | 128 scans |
Dummy Scans: | 8 |
Acquisition Time: | 4 s |
Relaxation Delay: | 1 s |
Spectral Width: | 9523.8 |
Num Data Points Acquired: | 38094 |
Real Data Points: | 65536 |
Line Broadening: | 0.22 Hz |
Zero Filling: | 65,536 points |
Apodization: | Exponential |
Baseline Correction Method: | Spline |
Chemical Shift Ref Std: | 0 ppm for DSS |
Binned Increment: | None |
Binned Data Excluded Range: | None |