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Status |
Public on Dec 10, 2021 |
Title |
3G-M_stimulated_replicate3 |
Sample type |
RNA |
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Source name |
Human T-cells expressing C-3 CAR, 24h stimulated on T47D CSF1R cells
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Organism |
Homo sapiens |
Characteristics |
stimulation: T47D CSF1R cells (stimulated) car type: 3rd generation chimeric co-stimulatory receptor (ccr): No car targeting moiety: CSF1 ccr targeting moiety: N/A car endodomain: CD28-41BB-CD3z ccr endodomain: N/A
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Treatment protocol |
CAR-T cells were labelled using the CellTrace CFSE Cell Proliferation Kit (ThermoFisher Scientific) according to the manufacturer's protocol and were stimulated on a confluent T47D or T47D CSF1R monolayer. After 24h stimulation, live (DAPI-), CFSE+ T-cells were flow sorted subsequently lysed for RNA isolation.
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Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation, activated with 5μg/mL phytohemagglutinin-L in RPMI supplemented with GlutaMax and 5% human serum for 48h, after which 100U/mL IL-2 was added for a further 24h. The activated T-cells were then transduced with retrovirus encoding the CAR constructs and a chimeric cytokine receptor which enabled expansion in IL-4. The T-cells were expaned for a further 7 days in the presence of 30 ng/mL IL-4 prior to antigen-specific stimulation.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using the Rneasy Mini Kit (Qiagen) according to the manufacturer's protocol.
|
Label |
N/A
|
Label protocol |
Labelled probes were provided by NanoString.
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Hybridization protocol |
For hybridisation 85ng RNA was used with the CAR-T Characterization Panel (XT-CSO-CART1) at 65C for 23 hours. This was followed by sample processing with the NanoString nCounter® Sprint Profiler (NanoString Technologies, Seattle, WA, USA) following the manufacturer's instructions.
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Scan protocol |
Sample scanning was performed with the Sprint profiler following the manufacturer's instructions.
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Data processing |
Raw data (RCC files) were used directly as input for the open source R package, NanoStringNorm for backgorund correction and between-sample normalization.
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Submission date |
Oct 26, 2021 |
Last update date |
Dec 10, 2021 |
Contact name |
John Maher |
E-mail(s) |
John.maher@kcl.ac.uk
|
Organization name |
King's College London
|
Street address |
Great Maze Pond
|
City |
London |
ZIP/Postal code |
SE1 9RT |
Country |
United Kingdom |
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|
Platform ID |
GPL29417 |
Series (1) |
GSE186557 |
Optimized delivery of dual co-stimulation and anti-tumor activity using parallel chimeric antigen receptors (pCARs) |
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