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MB Sample ID: SA091199
| Local Sample ID: | Patrick Media 16 |
| Subject ID: | SU001320 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001320 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Patrick Media 16 | SA091199 | FL013126 | cPLA2α KO | Genotype |
| Patrick Media 16 | SA091199 | FL013126 | Unscratched | Treatment |
Collection:
| Collection ID: | CO001314 |
| Collection Summary: | pDFs obtained from WT, KI, and KO mice were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 ul pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h. |
| Sample Type: | Fibroblasts |
Treatment:
| Treatment ID: | TR001335 |
| Treatment Summary: | Primary Dermal Fibroblasts were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 l pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h. |
Sample Preparation:
| Sampleprep ID: | SP001328 |
| Sampleprep Summary: | Samples were stored at − 80 °C until the time of analysis. Lipids were extracted for eicosanoids. Eicosanoid Preparation Eicosanoids were extracted using a modified extraction process from previously described. (1, 2). 4 mL of media was combined with an IS mixture comprised of comprised of 10% methanol (400 μl), glacial acetic acid (20 μl), and internal standard (20 μl) containing the following deuterated eicosanoids (1.5 pmol/μl, 30 pmol total) (All standards purchased from Cayman Chemicals): (d4) 6keto-Prostaglandin F1α, (d4) Prostaglandin F2α, (d4) Prostaglandin E2, (d4) Prostaglandin D2, (d8) 5-Hydroxyeicosa-tetranoic acid (5-HETE), (d8) 12-Hydroxyeicosa-tetranoic acid (12-HETE) (d8) 15-Hydroxyeicosa-tetranoic acid (15-HETE), (d6) 20-Hydroxyeicosa-tetranoic acid (20-HETE), (d11) 8,9 Epoxyeicosa-trienoic acid, (d8) 14,15 Epoxyeicosa-trienoic acid, (d8) Arachidonic acid, (d5) Eicosapentaenoic acid, (d5) Docosahexaenoic acid, (d4) Prostaglandin A2, (d4) Leukotriene B4, (d4) Leukotriene C4, (d4) Leukotriene D4, (d4) Leukotriene E4, (d5) 5(S),6(R)-Lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso Prostaglandin F2α, (d11) (±)14,15-DHET, (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) Prostaglandin E1, (d4) Thromboxane B2, (d6) dihmo gamma linoleic acid, (d5) Resolvin D2, (d5) Resolvin D1, (d5) Maresin 2, and (d5) Resolvin D3. Samples and vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH2O (50:50;100 μl) prior to UPLC ESI-MS/MS analysis. |
Combined analysis:
| Analysis ID | AN002078 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 |
| Column | Acentis Express C18 column |
| MS Type | MALDI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | NEGATIVE |
| Units | pmol of lipid |
Chromatography:
| Chromatography ID: | CH001515 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Acentis Express C18 column |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001929 |
| Analysis ID: | AN002078 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | MALDI |
| MS Comments: | Sciex Analyst software was used for analysis |
| Ion Mode: | NEGATIVE |
