|
| Status |
Public on Dec 31, 2022 |
| Title |
NK cells_expanded_replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
purified human NK cells, after 14 days expansion
|
| Organism |
Homo sapiens |
| Characteristics |
number of pooled donors: 7
stimulation: after 14 days expansion
sample pairs: pair3
|
| Treatment protocol |
Isolated primary NK cells were either directly used for further analysis or expanded for 14 days with cell sized magnetic particles loaded with CD2 and CD335 (NK Cell Activation and Expansion kit, Miltenyi Biotec). SCGM (Cell Genix) supplemented with 1000U/mL IL-2 (Proleukin, Novartis) and 5% AB Serum (Lonza) was used as cultivation medium.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMC) were obtained from Buffy Coat of healthy donors by density gradient centrifugation (Ficoll, GE Healthcare) followed by untouched NK cell isolation using the NK Cell Isolation Kit (Miltenyi Biotec). Alternatively NK cells were purified using MACSxpress NK Cell Isolation Kit enabling direct isolation without prior PBMC preparation (Miltenyi Biotec). For microarray analysis, isolated primary cells or expanded cells from 5-7 donors were pooled (n=4, total number of donors: 24). From this pool, we cryopreserved 1x107 cells for microarray analysis and flow cytometry.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using NucleoSpin® RNA kit (Macherey & Nagel) following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
100 ng of each RNA was used as template to produce Cy3- labeled cRNA. The RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Cy3 labeled cRNAs were hybridized overnight (17 hours, 65°C) to Agilent Whole Human Genome Oligo Microarrays (8x60K v1, Design ID 028004) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min and a final wash step with acetonitrile for 30 sec at RT.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent Technologies, Palo Alto, USA).
|
| Description |
gene expression analysis of T7-amplified polyA RNA
|
| Data processing |
The Agilent Feature Extraction Software (FES 10.5.1.1 ) was used to read out and process the microarray image files.
|
| |
|
| Submission date |
Jan 09, 2015 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Stefan Tomiuk |
| E-mail(s) |
stefant@miltenyibiotec.de
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Bioinformatics
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch-Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE64812 |
Analysis of Primary and Expanded NK Cells for Future Clinical Use |
|
