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MB Sample ID: SA343065
| Local Sample ID: | 5 |
| Subject ID: | SU003288 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003288 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 5 | SA343065 | FL040528 | MCJ KO CART in vivo | Factor |
| 5 | SA343065 | FL040528 | T-Cells | Sample source |
Collection:
| Collection ID: | CO003281 |
| Collection Summary: | The E2a cells (106) were cultured in CMM for no more than 2 passages before intravenous tail vail injection to B6 mice 3 days before CAR-T cell injection. The E2a-bearing mice were irradiated with 500cGy sublethal dosage the day before CAR-T cell injection. The murine CD8 CAR-T cells were generated and expanded with rhIL-2 (60 IU/ml) as described above. The expanded CD8 CAR-T cells were washed with PBS, and 106 CAR+ cells were injected into the E2a-bearing mice via intravenous tail vail injection. The mice were followed for survival with the humane end point of development of hind limb paralysis, or weight loss of more than 20% of the original weight, or signs of major discomfort as determined by the institutional veterinarian. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR003297 |
| Treatment Summary: | Metabolomics differences in this experiment are observed at baseline |
Sample Preparation:
| Sampleprep ID: | SP003295 |
| Sampleprep Summary: | CAR-T cells were isolated as described above either from in vitro culture or from bone marrow harvested in an in vivo study. The cells were washed in PBS and frozen at -80C until the assay is ready to run. Metabolites from cells were extracted at 2x106 cells/ml at 4°C (30 min) in the presence of 5:3:2 MeOH:MeCN:water (v/v/v). The samples were spun down and the resulting supernatant was transferred to new tubes and dried under a vacuum. The resulting residue was reconstituted in 0.1% formic acid at a 3x concentration, then analyzed on a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive MS as previously described in detail. |
Combined analysis:
| Analysis ID | AN005200 | AN005201 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Exploris120 | Exploris120 |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | Peak Areas | Peak Areas |
Chromatography:
| Chromatography ID: | CH003934 |
| Chromatography Summary: | Negative ion Mode |
| Instrument Name: | Exploris120 |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 95% water/5% acetonitrile; 1 mM ammonium acetate |
| Solvent B: | 95% acetonitrile/5% water; 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003935 |
| Chromatography Summary: | Positive Ion Mode |
| Instrument Name: | Exploris120 |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B. |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004933 |
| Analysis ID: | AN005200 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004934 |
| Analysis ID: | AN005201 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | POSITIVE |
