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MB Sample ID: SA057657
| Local Sample ID: | DS_38 |
| Subject ID: | SU001002 |
| Subject Type: | Mammal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Genotype Strain: | Fisher rat |
| Age Or Age Range: | 10-week-old |
| Weight Or Weight Range: | not measured |
| Height Or Height Range: | not measured |
| Gender: | Male |
| Animal Animal Supplier: | Charles River |
| Animal Housing: | standard |
| Animal Light Cycle: | standard 12 h |
| Animal Feed: | standard grain-based |
| Animal Water: | standard unlimited |
| Animal Inclusion Criteria: | random |
| Species Group: | - |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001002 |
| Subject Type: | Mammal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Genotype Strain: | Fisher rat |
| Age Or Age Range: | 10-week-old |
| Weight Or Weight Range: | not measured |
| Height Or Height Range: | not measured |
| Gender: | Male |
| Animal Animal Supplier: | Charles River |
| Animal Housing: | standard |
| Animal Light Cycle: | standard 12 h |
| Animal Feed: | standard grain-based |
| Animal Water: | standard unlimited |
| Animal Inclusion Criteria: | random |
| Species Group: | - |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| DS_38 | SA057657 | FL010375 | naïve control | Treatment |
Collection:
| Collection ID: | CO000996 |
| Collection Summary: | Experimental groups consisted of naïve controls (control), ON crush + intravitreal Zymosan + CPT-cAMP (regeneration), and ON crush + intravitreal vehicle (crush). Retinas were harvested on day 7 and 14 and ON were harvested on day 3, 7 and 14 post-crush. |
| Collection Protocol Comments: | retina and optic nerves collected |
| Sample Type: | Eye tissue |
| Collection Frequency: | day 3, 7 and 14 post-crush |
| Storage Conditions: | -80℃ |
| Collection Vials: | 1.5 mL polypropylene tubes |
| Storage Vials: | 1.5 mL polypropylene tubes |
Treatment:
| Treatment ID: | TR001016 |
| Treatment Summary: | A rat model of inflammation-induced ON regeneration was established by intravitreal injection of a yeast cell wall preparation (Zymosan A) and a cell permeant CPT-cAMP, immediately after ON crush. Ten-week-old male Fischer rats were deeply anesthetized with inhaled isoflurane, and the eyes were treated with topical anesthetic (proparacaine HCl 0.5% ophthalmic) and a cycloplegic (tropicamide 0.5% ophthalmic) to reduce pain and assist with visualization of intravitreal injections. The left ON was exposed by blunt dissection through a temporal, fornix-based conjunctival incision and crushed for 10 seconds with Dumoxel #N5 self-closing forceps (Dumont, Montignez, Switzerland). Absence of injury to the retinal vascular supply was confirmed by post-crush funduscopic examination. Intravitreal injections (5 µL) of PBS vehicle or a suspension of finely ground, sterilized Zymosan A (Z4250; Sigma-Aldrich, St. Louis, MO, USA; 12.5 mg/mL) plus CPT-cAMP (C3912; Sigma-Aldrich, St. Louis, MO, USA; 100 µM) were performed with a pulled glass pipette attached to a Hamilton syringe on a manual micromanipulator. Injections were made 2 mm posterior to the limbus, and care was taken to prevent lens injury, choroidal hemorrhage, or retinal detachment. Absence of these intraocular adverse events was confirmed by fundoscopic examination. Conjunctival incisions were closed with 8-0 vicryl sutures and petrolatum ophthalmic ointment was applied to the ocular surface. |
Sample Preparation:
| Sampleprep ID: | SP001009 |
| Sampleprep Summary: | Specimens were stored at -80ºC. Before extraction, ON and retinas were cut into ~1 mm3 pieces. 6 mL of methanol (LC-MS grade) and 3 mL of chloroform (LC-MS grade) were added to each sample. For this high throughput approach, no internal standards were added to samples. After 2 min vigorous vortexing and 2 min sonication in ultrasonic bath, the samples were incubated at 48ºC overnight (in borosilicate glass vials, PTFE-lined caps). The following day, 3 mL of water (LC-MS grade) and 1.5 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4ºC for 15 min to obtain phase separation. Lower phases were collected and dried in a centrifugal vacuum concentrator. Samples were stored at -20ºC until reconstituted in 60 µL of chloroform:methanol (1:1) prior to mass spectrometric analysis. |
| Sampleprep Protocol Filename: | method.docx |
| Processing Method: | lipid extraction |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | -20℃ |
| Sample Spiking: | no internal standards added |
Combined analysis:
| Analysis ID | AN001577 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Accela 600 |
| Column | Thermo Acclaim C30 (150 x 2.1mm,3um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH001106 |
| Chromatography Summary: | Reversed phase chromatographic separation was achieved using Accela Autosampler, Accela 600 pump and Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo Fisher Scientific, Waltham, MA). The column temperature was maintained at 30 ºC (negative mode) or 45ºC (positive mode) and tray temperature at 20ºC. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid (LC-MS grade). Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% formic acid. The flow rate was 260 µL/min and injection volume was 5 µL. The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min. |
| Instrument Name: | Thermo Accela 600 |
| Column Name: | Thermo Acclaim C30 (150 x 2.1mm,3um) |
| Column Temperature: | 30 (negative mode)/45 (positive mode) |
| Flow Gradient: | The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min. |
| Flow Rate: | 260 µL/min |
| Sample Injection: | 5 µL |
| Solvent A: | 60% methanol/40% water; 0.2% formic acid; 10 mM ammonium acetate |
| Solvent B: | 60% methanol/40% chloroform; 0.2% formic acid; 10 mM ammonium acetate |
| Analytical Time: | 20 min |
| Oven Temperature: | 20ºC |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001455 |
| Analysis ID: | AN001577 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | UNSPECIFIED |
| Capillary Temperature: | 310ºC (negative mode) or 350ºC (positive mode) |
| Collision Energy: | 40±30% for the negative mode and 30, parallel with 19±5% for the positive mode |
| Ionization: | +/- |
| Spray Voltage: | 4.4 kV |
| Automatic Gain Control: | MS: 1x106; MS/MS: 2x105 (negative mode) or 1x105 (positive mode) |
| Dataformat: | .RAW |
| Resolution Setting: | MS: 70,000; MS/MS: 17,500 |
