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MB Sample ID: SA343039
| Local Sample ID: | D16 19BBz_4 |
| Subject ID: | SU003286 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003286 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| D16 19BBz_4 | SA343039 | FL040523 | low 19BBz | Factor |
| D16 19BBz_4 | SA343039 | FL040523 | T-Cells | Sample source |
Collection:
| Collection ID: | CO003279 |
| Collection Summary: | Human CD8 cells were isolated with positive selection as described. The CD8 cells were activated with anti-CD3/anti-CD28 beads (Dynabeads™ Human T-Expander CD3/CD28, Gibco), as previously described107. After 48h activation, the CD8 cells were spun (1000xg for 2h at 32C) with lentiviral supernatant containing CD19-BBz/shMCJ-1 CAR, CD19-BBz/shMCJ-2 CAR or CD19-BBz/shMCJ-2 CAR construct-packing virus with rhIL-2 (40 IU/ml) and protamine sulfate. After transduction, the anti-CD3/anti-CD28 beads were removed and CD8 cells were expanded with rhIL-2 (100 IU/ml) for the specified number of days. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR003295 |
| Treatment Summary: | The human CD19-BBz shRNA CAR lentiviral constructs were based on a previously described CD19-BBz CAR containing the human CD19-binding scFV FMC63, CD8 hinge domain, 4-1BB costimulatory domain and CD3 chain105. Using CD19-BBz CAR plasmid as a cloning vector, we generated multiple vectors where we incorporated the RNA polymerase III U6 promotor (on the 3' of the CD3 chain domain) followed by an shRNA: 1) a CD19-BBz/shMCJ-1 CAR construct containing the shMCJ-1 5’-GAAGATTTCAACTCCTAGC-3’ sequence106, 2) a CD19-BBz/shMCJ-2 CAR construct containing the shMCJ-2; 5’-AACCTCTAGAACAAGTTATC-3’, and 3) a CD19-BBz/c-shRNA CAR vectors expressing the shRNA encoding scramble sequences. Lentiviral supernatant was produced in the LentiX-293T packaging cell line (Clonetech) as previously described. Lentiviral supernatants were collected after 48 hours post-transfection. |
Sample Preparation:
| Sampleprep ID: | SP003293 |
| 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 | AN005196 | AN005197 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Exploris120 | Exploris120 |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | Peak Areas | Peak Areas |
Chromatography:
| Chromatography ID: | CH003931 |
| Chromatography Summary: | Negative ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.2 min 5% B, 0.2-.8 min hold at 95% B, .8-.81 min 95-5% B, .81-1 min hold at 5% B. |
| Flow Rate: | 0.450 ml/min |
| Solvent A: | 100% Water; 10mM Ammonium acetate, .01% Formic Acid |
| Solvent B: | 50% Methanol/ 50% Acetonitrile; 10mM Ammonium acetate, .01% Formic Acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003932 |
| Chromatography Summary: | Positive Ion Mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.3 min 5% B, 0.3-.8 min hold at 95% B, .8-.81 min 95-5% B, .81-1 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: | MS004929 |
| Analysis ID: | AN005196 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Exploris120 |
| 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: | MS004930 |
| Analysis ID: | AN005197 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Exploris120 |
| 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 |
