|
| Status |
Public on Dec 10, 2021 |
| Title |
pCAR-M/34_unstimulated_replicate3 |
| Sample type |
RNA |
| |
|
| Source name |
Human T-cells expressing p34BB/C CAR, 24h stimulated on T47D cells
|
| Organism |
Homo sapiens |
| Characteristics |
stimulation: T47D cells (control)
car type: 2nd generation
chimeric co-stimulatory receptor (ccr): Yes
car targeting moiety: CSF1
ccr targeting moiety: IL-34
car endodomain: CD28-CD3zeta
ccr endodomain: 41BB
|
| 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.
|
| 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.
|
| 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.
|
| |
|
| 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.
|
| Scan protocol |
Sample scanning was performed with the Sprint profiler following the manufacturer's instructions.
|
| 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.
|
| |
|
| 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 |
| |
|
| Platform ID |
GPL29417 |
| Series (1) |
| GSE186557 |
Optimized delivery of dual co-stimulation and anti-tumor activity using parallel chimeric antigen receptors (pCARs) |
|
