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MB Sample ID: SA186803
| Local Sample ID: | 8h 2+ |
| Subject ID: | SU002078 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002078 |
| 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 |
|---|---|---|---|---|
| 8h 2+ | SA186803 | FL023113 | 8h engulfing | category |
Collection:
| Collection ID: | CO002071 |
| Collection Summary: | Experimental animals This study was approved and performed in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee at National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO (004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with DsRed mice until double homozygous. Mice used in experiments were between 8-16 weeks of age. Both male and female sex were used in experiments except bone marrow chimera experiments, which used only male mice. Human tissue This study used deidentified donor peripheral blood mononuclear cells (PBMC) collected and isolated (Young et al., 2011) by the National Jewish Blood Prep Core (IRB No. HS-1285). Age and sex of deidentified blood donors in this study is not known. Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat cell line was established from peripheral blood of a 14-year-old male diagnosed with T cell leukemia. Murine primary cell cultures Peritoneal cells were isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640 containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well plates for FACS sorting or 4x106 macrophage/well in 24-well plates for flow cytometry. Non-adherent cells were removed by washing after 3-5 hours, and macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli O111:B4 LPS (List Biological Laboratories Inc.) was added to generate inflammatory macrophages 12h before the addition of target cells. Bone marrow-derived macrophages were generated by 8-day culture of murine bone marrow cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS, 20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100M Sodium Pyruvate, 55M beta-Mercaptoethanol). All macrophages were removed from culture dishes by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping. |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR002090 |
| Treatment Summary: | LPS-primed murine peritoneal macrophages were fed fluorescently-tagged IgG-opsonized live Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 3 or 7 hours. Macrophages were then collected and sorted into non-engulfing or engulfing populations, alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were snap frozen and stored at -80C until metabolite extraction. |
Sample Preparation:
| Sampleprep ID: | SP002084 |
| Sampleprep Summary: | To process cells for assessment of intracellular metabolites, cells were pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and discarded; residual liquid was carefully wicked away from the pellet with a kimwipe. Dry pellets were immediately snap frozen and stored at -80C until processing. To process culture supernatants for assessment of metabolites, supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant was then transferred to a fresh tube, snap frozen, and stored at -80C until processing. |
Combined analysis:
| Analysis ID | AN003257 | AN003258 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002401 |
| Chromatography Summary: | Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile) at 250 uL/min. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| Column Temperature: | 25 |
| Flow Gradient: | Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile) at 250 uL/min. |
| Flow Rate: | 250 ul/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002402 |
| Chromatography Summary: | Isocratic flow of 100% phase A (95% water, 5% acetonitrile, 10 mM ammonium acetate) at 250 uL/min. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| Column Temperature: | 25 |
| Flow Gradient: | isocratic |
| Flow Rate: | 250 ul/min |
| Solvent A: | 95% water/5% acetonitrile; 10 mM ammonium acetate |
| Solvent B: | N/A |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003029 |
| Analysis ID: | AN003257 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017). |
| Ion Mode: | POSITIVE |
| MS ID: | MS003030 |
| Analysis ID: | AN003258 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017). |
| Ion Mode: | NEGATIVE |
