Return to study ST002551 main page
MB Sample ID: SA256210
Local Sample ID: | 180968_2_GREENL_ONC_OS_7dpi_POS |
Subject ID: | SU002651 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002651 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
180968_2_GREENL_ONC_OS_7dpi_POS | SA256210 | FL032763 | Crush | Treatment |
Collection:
Collection ID: | CO002644 |
Collection Summary: | For tissue collection, animals were euthanized using CO2 and the optic nerve was removed by dissection from the optic nerve head to the optic chiasm. |
Sample Type: | Eye tissue |
Treatment:
Treatment ID: | TR002663 |
Treatment Summary: | The C57BL/6 mice were injected with AAV2-CNTF at titer of 1x10e13 vg/ml (viral genomes per mL) with 1 uL of AAV-CNTF injected in each eye. Mice labeled as AAV2-Green-Lantern were injected as a control. Prior to surgery, mice received a cocktail of ketamine and xylazine for anesthesia. The optic nerve crush was performed by exposing the optic nerve by blunt dissection. The nerve was crushed using forceps for 10 seconds 1 mm behind the globe. At 7 and 14 days post crush mice were euthanized and optic nerves were collected. |
Sample Preparation:
Sampleprep ID: | SP002657 |
Sampleprep Summary: | Metabolite extraction was carried out while keeping the optic nerve tissues on dry ice to prevent degradation. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Add 84 µL of chilled 1:1 MeOH/H2O to Precellys tube. Pre-extraction internal standards were added to the tubes. Add pre-extraction internal standards: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, and 1µl of 5mg/mL Isoleucine 13C6 to each sample. Tissues were homogenized using Precellys 24 Touch. Cycle parameters: 2 cycles: 30 seconds homogenization at 4500 rpm, 10 seconds rest. Transfer homogenate to microcentrifuge tube and centrifuge at 18000xrcf for 20 min at 4°C. Collect supernatant and transfer pellet to Precellys Lysing Kit tube. Add 84uL of 8:1:1 Acetonitrile/Methanol/Acetone to pellet and add the rest of the pre-extraction internal standards: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, 1µl of 5mg/mL Isoleucine 13C6. Final pre-extraction internal standards concentrations are 50μg/mL. Homogenization cycles were repeating using Precellys 24 Touch. Centrifuge as before and add second supernatant to first round of collected supernatant. Centrifuge at 1800xrcf for 20 min once more to remove any remaining tissue debris. Collect supernatant and dry supernatant in Speedvac. Two extraction blanks were prepared in the same manner as the biological samples. Dried samples were reconstituted immediately in 0.1% formic acid in 44.75µL of HPLC-MS grade water. Post-extraction internal standards were added: 25 µl of 5mg/ml Phenylalanine 13C6, 2.5 µl of .5mg/ml Uracil 13C 15N2, 1.25 µl of 1mg/ml Arginine 13C6, 1.25 µl of 1mg/ml Serine 13C3 to each sample. |
Combined analysis:
Analysis ID | AN004200 | AN004201 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Vanquish | Thermo Vanquish |
Column | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | µg/ml | µg/ml |
Chromatography:
Chromatography ID: | CH003113 |
Chromatography Summary: | Mobile Phases: NEG A: 10mM Ammonium Acetate in 95% ACN w/ .1% acetic acid NEG B: 10mM Ammonium Acetate in 50% ACN w/ .1% acetic acid POS A: 10mM Ammonium Formate in 95% ACN w/ .1% formic acid POS B: 10mM Ammonium Formate in 50% ACN w/ .1% formic acid |
Instrument Name: | Thermo Vanquish |
Column Name: | Thermo Accucore Amide HILIC (150 x 2.1mm, 2.6um) |
Column Temperature: | 35 |
Flow Gradient: | PumpModule.Pump.Pump_Pressure.AcqOn 0.000 [min] Run PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 1.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 9.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 95.0 [%] PumpModule.Pump.Curve: 5 10.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 95.0 [%] PumpModule.Pump.Curve: 5 10.500 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 15.000 [min] PumpModule.Pump.Flow.Nominal: 0.500 [ml/min] PumpModule.Pump.%B.Value: 1.0 [%] PumpModule.Pump.Curve: 5 15.000 [min] Stop Run |
Flow Rate: | 0.5 ml/min |
Solvent A: | 95% acetonitrile/5% water; 10mM Ammonium Formate; 0.1% formic acid |
Solvent B: | 50% acetonitrile/50% water; 10mM Ammonium Formate; 0.1% formic acid |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003947 |
Analysis ID: | AN004200 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The samples were run using a Q ExactiveTM mass spectrometer coupled to a heated electrospray ionization (HESI) source. The spray voltage was set to 3.50 kV, capillary temperature to 350°C, sheath gas to 55, aux gas to 14, sweep gas to 4, and S-Lens RF Level to 30.0. The mass range was set to 67 – 1000 m/z, resolution 140,000 for full scan and 35,000 for ddMS2. AGC target was set to 1e6 for full scan and 2e5 for ddMS2. The max injection time (IT) was 100 seconds for full scan mode and 50 seconds for ddMS2. The number of microscans was 2, and normalized collision energy (NCE) was set to 20, 35, and 50. Samples were run in both positive and negative ion mode separately. The parameters for negative mode were the same except the spray voltage, which was set to 2.50 kV and capillary temperature to 380°C. Metabolites were identified from their Thermo.RAW scans using Compound DiscovererTM 3.3 software. Extraction blanks were used to determine and correct for reagent effects, allow for the creation of exclusions lists, mark background components, and filters the background components from the results table in Compound DiscovererTM 3.3. Pooled QCs were used for initial compound normalization and identification. All non-identified compounds were removed. |
Ion Mode: | POSITIVE |
MS ID: | MS003948 |
Analysis ID: | AN004201 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The samples were run using a Q ExactiveTM mass spectrometer coupled to a heated electrospray ionization (HESI) source. The spray voltage was set to 3.50 kV, capillary temperature to 350°C, sheath gas to 55, aux gas to 14, sweep gas to 4, and S-Lens RF Level to 30.0. The mass range was set to 67 – 1000 m/z, resolution 140,000 for full scan and 35,000 for ddMS2. AGC target was set to 1e6 for full scan and 2e5 for ddMS2. The max injection time (IT) was 100 seconds for full scan mode and 50 seconds for ddMS2. The number of microscans was 2, and normalized collision energy (NCE) was set to 20, 35, and 50. Samples were run in both positive and negative ion mode separately. The parameters for negative mode were the same except the spray voltage, which was set to 2.50 kV and capillary temperature to 380°C. Metabolites were identified from their Thermo.RAW scans using Compound DiscovererTM 3.3 software. Extraction blanks were used to determine and correct for reagent effects, allow for the creation of exclusions lists, mark background components, and filters the background components from the results table in Compound DiscovererTM 3.3. Pooled QCs were used for initial compound normalization and identification. All non-identified compounds were removed. |
Ion Mode: | NEGATIVE |