|
| Status |
Public on Jan 01, 2013 |
| Title |
HaCaT_Cyt+Ino_Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HaCaT cells, with cytokines and Ino-C2-PAF, 24h, replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HaCaT
treatment protocol: stimulated with cytokines (each 2 ng/ml) and 5 µM Ino-C2-PAF
|
| Treatment protocol |
The assay was performed in defined keratinocytes serum-free medium without growth supplement. Cells were treated with IL-1α, IL-17, IL-22, TNF-α and oncostatin-M (2 ng/ml each) in the presence or absence of 5 µM Ino-C2-PAF or left untreated (control) 24 h.
|
| Growth protocol |
HaCaT cells were grown in RPMI medium supplemented with heat-inactivated fetal bovine serum (10 %), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamine (440 mg/l). One day prior to experimentation, cells were adapted to defined keratinocyte serum-free medium with growth supplement (including insulin, EGF, and FGF).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit in accordance to the manufacturer instructions (Qiagen Hilden, Germany). The integrity of RNA was verified by the presence of the 28S and 18S rRNA on agarose gels and an A260/280 ratio in the range of 1.9–2.1.
|
| Label |
Cy3
|
| Label protocol |
200 ng of total RNA were used for production of fluorescent (Cyanine-3) cRNA as described in the Agilent analysis instruction manual for One-Color Microarray-Based Gene Expression Analysis (Agilent Technologies, Palo Alto, USA). Amplified cRNA was purificated using RNAeasy mini spin columns and subsequently quantified using NanoDrop ND-1000 UV-VIS Spectrophotometer.
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| |
|
| Hybridization protocol |
All samples were hybridized to Agilent whole human genome microarray kit 8x60K (G4851A) according to the manufacturer´s instructions (One-Color Microarray-Based Gene Expression Analysis).
|
| Scan protocol |
Arrays were scanned at 5 µm resolution on an Agilent DNA Microarray Scanner (G2505C, Agilent).
|
| Description |
Gene expression after 24h HaCaT cells stimulated with cytokines (each 2 ng/ml) and 5 µM Ino-C2-PAF
|
| Data processing |
The signal values were extracted using the Agilent Feature Extracting Software version 10.7.3.1 and protocol GE_107_Sep09.
|
| |
|
| Submission date |
Apr 17, 2012 |
| Last update date |
Jan 01, 2013 |
| Contact name |
Geo Semini |
| E-mail(s) |
geosemini@yahoo.com
|
| Organization name |
Charité - Universitätmedizin
|
| Department |
Institute for Biochemie
|
| Street address |
Oudenarderstrasse 16
|
| City |
Berlin |
| ZIP/Postal code |
13347 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE37361 |
Impact of Ino-C2-PAF on the gene expression profile of HaCaT cells after stimulation with pro-inflammatory cytokines |
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