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MB Sample ID: SA343027
| Local Sample ID: | MCJ KO CD19-BBz CART_3 |
| Subject ID: | SU003285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| MCJ KO CD19-BBz CART_3 | SA343027 | FL040520 | MCJ KO | factor |
| MCJ KO CD19-BBz CART_3 | SA343027 | FL040520 | T-Cells | Sample source |
Collection:
| Collection ID: | CO003278 |
| Collection Summary: | Mouse CD8 cells were isolated with negative selection as described above, activated with anti-CD3/anti-CD28 beads (Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation, Gibco, Cat#11453D) with addition of recombinant human IL-2 (rhIL-2) (60 IU/ml) and human recombinant IL-7 (10 ng/ml). On the second and third day of activation, the supernatant containing the CD19-BBz CAR/hEGFR retrovirus was spun on a plate coated with retronectin (Takara Bio Inc.) (2000xg for 2.5h at 32C). The activated cells were added to the viral-coated plate for transduction. The transduced CD8 CAR-T cells were removed from the beads on the fourth day and expanded with rhIL-2 (60 IU/ml) or rhIL-7 (10 ng/ml) and rhL-15 (100 ng/ml). After 2 days (1st expansion) cells were split 1/3 with fresh cytokine-containing medium. After 2 more days (2nd expansion), cells were split again with fresh cytokine-containing medium. Two days later (3rd expansion) cells were used for experiment or incubated in cytokine free-medium. For CAR+ cells enrichment, the CAR-T cells expanded with IL-2 for 3 expansions were incubated with an anti-hEGFR-PE (BioLegend, Cat#352904, RRID: AB_10896794) antibody, followed by incubation with anti-PE microbeads (Miltenyi, order#130-048-801), and purified using the Miltenyi LS columns as recommended by the manufacturer (Miltenyi). The enrichment resulted in a 98-100% CAR+ population, as verified by flow cytometry. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR003294 |
| Treatment Summary: | Metabolomics differences in this experiment are observed at baseline |
Sample Preparation:
| Sampleprep ID: | SP003292 |
| 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 | AN005194 | AN005195 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| 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 Area | Peak Area |
Chromatography:
| Chromatography ID: | CH003929 |
| Chromatography Summary: | Negative ion Mode |
| Instrument Name: | Thermo Vanquish |
| 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: | CH003930 |
| Chromatography Summary: | Positive Ion Mode |
| Instrument Name: | Thermo Vanquish |
| 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: | MS004927 |
| Analysis ID: | AN005194 |
| 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: | MS004928 |
| Analysis ID: | AN005195 |
| 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 |
