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MB Sample ID: SA078002
| Local Sample ID: | 56 |
| Subject ID: | SU001185 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001185 |
| 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 |
|---|---|---|---|---|
| 56 | SA078002 | FL011870 | DMSO | Treatment |
| 56 | SA078002 | FL011870 | 12 | Time |
Collection:
| Collection ID: | CO001179 |
| Collection Summary: | Immediately following treatment, cell monolayers were washed 3 times with cold PBS, and cells were scraped and fixed in 500 μL 70% MeOH. Samples were stored immediately at -80°C until pretreatment for solid phase extraction (SPE), as follows. |
| Collection Protocol Filename: | AKB_Targeted-protocol.docx |
| Collection Protocol Comments: | For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file. |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR001200 |
| Treatment Summary: | The C10 cell line was obtained from Dr. Lori Nield (University of Colorado). These cells are an immortalized, non-transformed alveolar type II cell line originally derived from a BALB mouse (Bentel et al. 1989; Malkinson et al. 1997). C10 cells were treated with or without p38 inhibitor (CAY10502) 24 h following serum deprivation with either DMSO control or 40 μM the binary LMW PAH mixture for 2, 4, 8, 12, or 24 hr and DMSO control with an n=4/treatment. |
| Treatment Protocol Filename: | AKB_Targeted-protocol.docx |
| Treatment Protocol Comments: | For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file. |
Sample Preparation:
| Sampleprep ID: | SP001193 |
| Sampleprep Summary: | Briefly, before SPE cleanup, the frozen samples were removed from the freezer and centrifuged at 14,000 RPM at 4°C for 10 minutes. The supernatant was removed, transferred to a new microcentrifuge tube, and spiked with 10 μL of 10 pg/μL internal standard solution (100 pg of each: 5(S)-HETE-d8, 8-iso-PGF2a-d4, 9(S)-HODE-d4, LTB4-d4, LTD4-d5, LTE4-d5, PGE2-d4, PGF2a-d9 and RvD2-d5 in ethanol). The supernatant was dried in a vacuum centrifuge and reconstituted with 1.0 mL of 10% methanol. The reconstituted extracts were loaded on a Strata-X 33 μm 30 mg/1maL SPE column (Phenomenex, Torrance, California, USA) preconditioned with 1.0 mL of methanol followed by 1.0 mL of water. The SPE column was washed with 10% methanol and then eluted directly into a reduced surface activity/maximum recovery glass autosampler vial with 1.0 mL of methyl formate. The methyl formate was evaporated completely from the vial with a stream of nitrogen and the SPE cartridge was then eluted with 1.0 mL of methanol directly into the same autosampler vial. The methanol was evaporated to dryness with a stream of nitrogen and the sample was reconstituted with 20 uL of ethanol. The samples were analyzed immediately or frozen at -70 °C until analysis. |
| Sampleprep Protocol Filename: | AKB_Targeted-protocol.docx |
| Sampleprep Protocol Comments: | For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file. |
Combined analysis:
| Analysis ID | AN001849 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 1260 |
| Column | Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Agilent 6490 QQQ |
| Ion Mode | NEGATIVE |
| Units | pg per 1.5 million cells |
Chromatography:
| Chromatography ID: | CH001326 |
| Instrument Name: | Agilent 1260 |
| Column Name: | Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001710 |
| Analysis ID: | AN001849 |
| Instrument Name: | Agilent 6490 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | none |
| Ion Mode: | NEGATIVE |
