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MB Sample ID: SA004283
| Local Sample ID: | SBEP_Metab_LPSactivated2 |
| Subject ID: | SU000103 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | RAW 264.7 |
| Cell Strain Details: | RAW 264.7 |
| Cell Primary Immortalized: | immortalized |
| Species Group: | Mammal |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000103 |
| Subject Type: | Animal cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | RAW 264.7 |
| Cell Strain Details: | RAW 264.7 |
| Cell Primary Immortalized: | immortalized |
| Species Group: | Mammal |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SBEP_Metab_LPSactivated2 | SA004283 | FL001223 | 100 ng/ml | Lipopolysaccharide Treatment |
Collection:
| Collection ID: | CO000086 |
| Collection Summary: | After stimulation, cells were washed twice with Dulbeccos PBS, scraped out and harvested into 15-mL centrifuge tubes. |
| Sample Type: | Cell |
| Collection Method: | After stimulation, cells were washed twice with Dulbeccos PBS, scraped out and harvested into 15-mL centrifuge tubes. |
| Collection Time: | 24 hours |
| Collection Vials: | 15-mL centrifuge tubes |
| Tissue Cell Quantity Taken: | All |
Treatment:
| Treatment ID: | TR000104 |
| Treatment Summary: | Macrophages (RAW 264.7 cells) grown for two day with suplimentation of 100ng | ml lipopolysaccharide | Macrophages (RAW 264.7 cells) grown for two days without suplimentation |
| Treatment Protocol Comments: | Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, then grown for 2 days and stimulated for 24 hours with 100 ng/mL of lipopolysaccharide diluted in fresh medium. A control culture was run in parallel by incubating for the same period of time with fresh medium only. Three biological replicates were performed per condition, and two dishes were used for each replicate. / Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, then grown for 2 days and stimulated for 24 hours with 100 ng/mL of lipopolysaccharide diluted in fresh medium. A control culture was run in parallel by incubating for the same period of time with fresh medium only. Three biological replicates were performed per condition, and two dishes were used for each replicate. |
| Treatment: | Abiotic |
| Treatment Compound: | lipopolysaccharide / fresh medium |
| Treatment Dose: | 100 ng/mL /-- |
| Treatment Vehicle: | fresh medium |
| Cell Growth Container: | 150 mm dishes |
| Cell Inoc Proc: | Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes |
| Cell Media: | Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum |
| Cell Harvesting: | After stimulation, cells were washed twice with Dulbeccos PBS, scraped out and harvested into 15-mL centrifuge tubes. |
Sample Preparation:
| Sampleprep ID: | SP000099 |
| Sampleprep Summary: | Suspentions softly centrifuged, buffer removed, amonium bicarbonate added, metabolites extraxted with chloroform/methanol (2:1, v/v), vortexed, centrifuged, aqueous layer dried in vacuum concentrator, derivatization with methoxyamine in pyridine, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and 1% trimethylchlorosilane (TMCS) |
| Sampleprep Protocol Comments: | Cell suspensions were softly centrifuged (230 × g for 5 min) and as much buffer as possible was removed. Then, 170 µL of 150 mM ammonium bicarbonate was added to the cell pellet and the cell suspensions were transferred to 2 mL micro-centrifuge tubes for extraction. Subsequently, the water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator. For the derivatization, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with shaking for 90 min to protect carbonyl groups. Next, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min to derivatize hydroxyl and amine groups. The samples were then allowed to cool to room temperature. |
| Processing Method: | Homogenization |
| Extraction Method: | The water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator. |
| Extract Concentration Dilution: | chilled (-20°C) chloroform/methanol (2:1, v/v) |
| Extract Enrichment: | Vacuum Concentrator |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS), |
| Cell Type: | Macrophage |
Combined analysis:
| Analysis ID | AN000136 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890A |
| Column | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5975C |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH000096 |
| Chromatography Summary: | Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation |
| Chromatography Comments: | Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min. |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent HP5-MS (30m × 0.25mm, 0.25 um) |
| Flow Rate: | 0.45-0.5mL/min |
| Injection Temperature: | 250 C |
| Sample Injection: | 1 L, Splitless |
| Analytical Time: | 37.5 min |
| Oven Temperature: | 60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C |
| Sample Syringe Size: | 10 L |
| Chromatography Type: | GC |
MS:
| MS ID: | MS000112 |
| Analysis ID: | AN000136 |
| Instrument Name: | Agilent 5975C |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis. |
| Ion Mode: | POSITIVE |
| Scan Range Moverz: | 50-550 m/z |
