Return to study ST002455 main page
MB Sample ID: SA245708
| Local Sample ID: | CO_Oxylipin_6 |
| Subject ID: | SU002544 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 6 weeks |
| Gender: | Female |
| Animal Animal Supplier: | JAX |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002544 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 6 weeks |
| Gender: | Female |
| Animal Animal Supplier: | JAX |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CO_Oxylipin_6 | SA245708 | FL030851 | Wild-type | Genotype |
| CO_Oxylipin_6 | SA245708 | FL030851 | LPS+antiPla2g5 | Treatment |
Collection:
| Collection ID: | CO002537 |
| Collection Summary: | C57BL/6J were injected intraperitoneally with 50 µg of anti-PLA2G5 neutralizing antibodies in 100 µl of PBS 1 hour prior to LPS injection. Twelve hours after LPS injection, mice were anesthetized with 2,2,2-tribromoethanol (250-500 mg/kg) and perfused transcardially with PBS containing 10 mM EDTA. Plasma was collected from blood prior to perfusion and frozen at -80°C. Immediately after perfusion, colon tissues were extensively washed in PBS and frozen by liquid nitrogen and kept at -80°C until the following procedures. |
| Sample Type: | Blood (plasma) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002556 |
| Treatment Summary: | C57BL/6J were injected intraperitoneally with 50 µg of anti-PLA2G5 neutralizing antibodies in 100 µl of PBS 1 hour prior to LPS injection. |
Sample Preparation:
| Sampleprep ID: | SP002550 |
| Sampleprep Summary: | Tissues were mechanically homogenized with the Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) in methanol containing internal standards (500 pmol/sample of d4-labeled EPA, d5-labeled PGE2, LPC with a 17:0 fatty acyl chain (LPC17:0), and PC with two 14:0 fatty acyl chains (PE14:0-14:0)) and then incubated overnight at -20°C. For extraction of phospholipids and lysophospholipids, one-tenth of tissue lysates were added to 10 volumes of 20 mM Tris-HCl (pH 7.4) and were extracted using the method of Bligh and Dyer. For extraction of oxygenated fatty acid metabolites, nine-tenths of the tissue lysates were added to water (final methanol concentration of 10% (v/v)), and the lipids were extracted using an Oasis HLB cartridge (Waters, Milford, MA, USA). |
Combined analysis:
| Analysis ID | AN004005 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 4000 QTrap |
| Ion Mode | UNSPECIFIED |
| Units | intensity |
Chromatography:
| Chromatography ID: | CH002958 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | linear |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | acetonitrile/methanol/water = 1/1/1 (v/v/v) containing 5 mM phosphoric acid and 1 mM ammonium formate |
| Solvent B: | 2-propanol containing 5 uM phosphoric acid and 1 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003753 |
| Analysis ID: | AN004005 |
| Instrument Name: | ABI Sciex 4000 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | The samples were applied to a Kinetex C18 column (Kinetex C18, 2.1 x 150 mm, 1.7 µm particle; Phenomenex, Inc., Torrance, CA, USA) connected with ESI-MS/MS on a liquid chromatography (NexeraX2 system; Shimadzu Co., Kyoto, Japan) coupled with a 4000Q-TRAP quadrupole-linear ion trap hybrid mass spectrometer (AB Sciex, Framingham, MA, USA). For analyses of free fatty acids (FFAs), lysophospholipids (LPLs) and phospholipids, the samples were applied to the column and separated by a step gradient with mobile phase A (acetonitrile/methanol/water =1/1/1 (v/v/v) containing 5 mM phosphoric acid and 1 mM ammonium formate) and mobile phase B (2-propanol containing 5 µM phosphoric acid and 1 mM ammonium formate) at a flow rate of 0.2 mL/min at 50°C. For analyses of oxygenated fatty acid metabolites, the samples were applied to the column and separated using a step gradient including mobile phase C (water containing 0.1 % acetic acid) and mobile phase D (acetonitrile/methanol = 4/1 (v/v)) at a flow rate of 0.2 mL/ min at 45°C. Identification of phospholipids, LPLs, FFAs, and oxygenated PUFAs (polyunsaturated fatty acids) metabolites was conducted by multiple reaction monitoring (MRM) transition, and quantification was performed based on the peak area of the MRM transition and the calibration curve obtained with an authentic standard for each compound. |
| Ion Mode: | UNSPECIFIED |
