|
| Status |
Public on Sep 25, 2023 |
| Title |
Donor_I_CD8_PD1_neg |
| Sample type |
RNA |
| |
|
| Source name |
Human abdominal skin
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Donor_I
age: 48
gender: Female
cell type: CD8_PD1_neg
cell sort and lysis: Following culture, cell population indicated in column A was FACS sorted and lysed in RLT at a concentration of ~1667 cells/ul
|
| Growth protocol |
Cultured human skin cells expanded for a total of 6 weeks in human IL-2 and IL15: 3mm punch biopsies obtained from discarded abdominal skin following elective plastic/reconstructive surgery were placed on tantalum coated grids and cultured in medium supplemented with human IL-2 and IL-15 for 3 weeks(Grid isolation). The cells were then taken 'off the grids' and cultured for a further 3 weeks in medium supplemented with human IL-2 and IL-15 (expansion phase).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were stained with a live dead exclusion dye and human antibodies specific for PD-1, CD3, TCRab, Vd1, CD8a and Pan gd. PD-1+ and PD-1- CD8 ab T cell and Vd1+ gd T cells were FACS sorted and lysed in RLT lysis buffer
|
| Label |
None
|
| Label protocol |
n/a
|
| |
|
| Hybridization protocol |
8 ul of lysed cells (except sample Donor_A_Vd1_PD1_pos where 4ul was run) were hybridised at 65 degrees celcius overnight to NanoString's human nCounter Immune exhaustion panel
|
| Scan protocol |
Hybridized samples were processed on an nCounter prep station (NanoString Technologies) following manufacturer's instructions.
|
| Description |
~13336 lysed cells in 8ul RLT buffer was hybridised to the nCounter panel at 65 degrees overnight.
|
| Data processing |
Data were collected on an nCounter digital analyzer (NanoString Technologies), following manufacturer’s instructions. Using nSolver 4.0 (NanoString Technologies), raw counts were scaled based on the geometric mean of all positive control probes. Gene expression normalization (normalised counts) was performed relative to the geometric mean of all housekeeping genes included in the panel. The lower limit of detection was set at a normalised count of 20 which encompassed all negative control reference probes.
|
| |
|
| Submission date |
Jul 20, 2023 |
| Last update date |
Sep 25, 2023 |
| Contact name |
Shraddha Kamdar |
| E-mail(s) |
shraddha.kamdar@kcl.ac.uk
|
| Organization name |
King’s College London
|
| Department |
Peter Gorer Department of Immunobiology
|
| Lab |
Immunosurveillance Lab
|
| Street address |
Guys Hospital,Great Maze Pond, London Bridge
|
| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL33412 |
| Series (1) |
| GSE232529 |
PD-1 defines a distinct, functional, tissue-adapted state in Vδ1+ T cells with implications for cancer immunotherapy |
|
