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MB Sample ID: SA236893
| Local Sample ID: | Glucose_Soleus_1D_DecON&OFF-2 |
| Subject ID: | SU002445 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 16±3 weeks |
| 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 chow diet food |
| Animal Water: | free access to food and water (3-5 animals per cage). |
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Subject:
| Subject ID: | SU002445 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 16±3 weeks |
| 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 chow diet food |
| Animal Water: | free access to food and water (3-5 animals per cage). |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Glucose_Soleus_1D_DecON&OFF-2 | SA236893 | FL029319 | Glucose_Soleus | Group |
Collection:
| Collection ID: | CO002438 |
| Collection Summary: | Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR analysis. |
| Collection Protocol Comments: | Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg tissue to afford detectable levels of substrates in the NMR analysis. |
| Sample Type: | Muscle |
| Collection Method: | Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. |
| Collection Location: | University of Florida, Applied Physiology and Kinesiology, MBI, 1149 Newell Dr, Gainesville, FL 32610 |
| Storage Conditions: | -80℃ |
| Collection Vials: | cryovials |
| Storage Vials: | cryovials |
Treatment:
| Treatment ID: | TR002457 |
| Treatment Summary: | Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for subsequent NMR analysis. N=4 muscles were pooled into a single biological replicate of 30-50 mg tissue to afford detectable levels of substrates in the NMR analysis. |
| Animal Vet Treatments: | none |
| Animal Anesthesia: | a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) |
| Animal Fasting: | non-fasted |
| Animal Endp Euthanasia: | Euthanasia was carried out by thoracotomy followed by cervical dislocation. |
| Animal Endp Tissue Coll List: | Skeletal muscle (soleus and EDL) |
Sample Preparation:
| Sampleprep ID: | SP002451 |
| Sampleprep Summary: | Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions were performed for all samples to isolate metabolites. The latter method was more efficient in sample recovery due to the reduced number of steps in the procedure but did not affect the proportion of metabolites. For PCA extraction, isolated muscle samples were homogenized with a FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) with 6% (v/v) ice cold PCA and centrifuged with 13.2 K rpm at 4 oC. The solid muscle portion was washed again with the 6% (v/v) ice cold PCA followed by centrifugation (13.2 K rpm) at 4 oC. The supernatant (combined) obtained was further neutralized with 5M potassium hydroxide and centrifuged again maintaining 13.2 K rpm speed at 4 oC. The resulting supernatants were then lyophilized (Thermo-Scientific, Dallas, USA). The pH of the dried powder was adjusted to 7.2 after dissolving it in 200 μL of ultra-pure water using 1M sodium hydroxide and 1 M hydrochloric acid. The pH-adjusted solution was further centrifuged, the resulting supernatant was dried and the powder was used to prepare the NMR sample. For acetonitrile:isopropanol:water extraction, homogenization of isolated muscle samples was carried out in 1 mL acetonitrile:isopropanol:water (3:3:2, v:v:v) ice cold mixture with a FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) and centrifuged at 4 oC in separate vials. Resultant supernatants were further lyophilized till dryness (Thermo-Scientific, Dallas, USA). The dried powder was further dissolved in 1 mL of Acetonitrile:Water (1:1, v:v) mixture, vortexed well for ~5 minutes. The resultant solution was further centrifuged, the supernatant obtained was further dried and the powder was used to prepare the NMR sample. The centrifugation speed for each step used was 13.2K rpm. Each NMR sample consisted of 50 mM phosphate buffer (pH 7), 2 mM EDTA, 0.02% of NaN3 with 0.5 mM of DSS as a standard internal reference in deuterated environment. 1H NMR spectra were taken at 25oC using a 600 MHz Bruker Avance II Console equipped with a TCI CryoProbe that utilized Bruker Topspin 4 software (Bruker BioSpin Corporation, Billerica, MA, USA). The first slice of a NOESY pulse sequence (noesypr1d) was used to acquire proton NMR. Fractional enrichment for glutamate, lactate and alanine were determined using 13C decoupling ON/OFF 1H proton spectra as well as 1D NOESY spectra. To determine enrichements, a standard zgig pulse sequence was adapted to allow 13C decoupling during the acquistion period (1.36 s) to remove the satellites. Total enrichment was measured by taking a ratio of the metabolite peak heights in the decoupling on/off experiments. NOESY spectra were collected with a 1 s relaxation delay (d1), and a 4 s acqusition time (at), in accordance with Chenomx recommendations for producing quantitative estimates of concentration. Using the Chenomx quantification and the fractional enrichments, a final concentration of the metabolites was calculated. Conventional 1H decoupled 13C spectra were acquired using a 600 MHz Agilent with a specially designed 1.5 mm superconducting (HTS) probe at 30oC. |
| Sampleprep Protocol Filename: | Isolated_muscle_Procedures.docx |
| Processing Method: | Lyophilization and Homogenization |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions |
| Extract Storage: | -80℃ |
| Sample Resuspension: | In 35 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: | SA236893 |
| Analysis ID: | AN003847 |
| Laboratory Name: | McKnight Brain Institute |
| Analysis Type: | NMR |
| Acquisition Date: | 11/17/2017 to 05/04/2021 |
| Software Version: | Topspin |
| Operator Name: | Ram Khattri |
| Detector Type: | Bruker 600 MHz |
| Data Format: | fid, 1r |
| Num Factors: | 4 |
| Num Metabolites: | 11 |
| Units: | mM |
NMR:
| NMR ID: | NM000255 |
| Analysis ID: | AN003847 |
| Instrument Name: | Bruker 600 MHz |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Field Frequency Lock: | Deuterium |
| Standard Concentration: | 0.5mM DSS |
| Spectrometer Frequency: | 600 MHz |
| NMR Probe: | 5 mm CPTXI 1H/D-13C/15N Z-GRD Z44866/0026 |
| 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 NMR tube |
| Shimming Method: | Topshim |
| Pulse Sequence: | zgig1d |
| Water Suppression: | none |
| Pulse Width: | 90-degree |
| Receiver Gain: | 25.4 |
| Offset Frequency: | 4.77 ppm |
| Chemical Shift Ref Cpd: | DSS |
| Temperature: | 298.2 oK |
| Number Of Scans: | 32 |
| Dummy Scans: | 8 |
| Acquisition Time: | 1.15s |
| Relaxation Delay: | 2.85 s |
| Spectral Width: | 7142.9 Hz |
| Num Data Points Acquired: | 32768 |
| 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 |
