|
| Status |
Public on Mar 14, 2017 |
| Title |
11_LPS-HFIP |
| Sample type |
RNA |
| |
|
| Source name |
HMVEC (CC-2815)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human microvascular endothelial cells (HMVEC)
exposed to: LPS 20mcg/mL and HFIP 8mmol/L over a time period of 6 hrs
|
| Biomaterial provider |
Lonza; CC-2815
|
| Treatment protocol |
Human microvascular endothelial cells (HMVEC, CC-2815) were stimulated with 20mcg/mL LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich, Buchs, Switzerland) to induce an inflammatory response. Hexafluoro-2-propanol (HFIP; Sigma-Aldrich, Buchs, Switzerland) was added to the cell culture medium containing LPS (Phosphate-buffered saline in controls) for a time period of 6 hours at a concentration of 8mmol/L.
|
| Growth protocol |
Human microvascular endothelial cells (CC-2815) derived from normal human microvascular blood vessels were purchased from Lonza (Verviers, Belgium) and cultured as described in Urner et. al., Am J Respir Cell Mol Biol. 2011;45(3):617-24.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from HMVEC was extracted using RNeasy (Qiagen) according to the manufacturer's protocol. The quality of the isolated RNA was evaluated using NanoDrop ND 1000 (NanoDrop Technologies, Delaware, USA) and Bioanalyzer 2100 (Agilent, Waldbronn, Germany).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green).
|
| Description |
Replicate 11 exposed to LPS and HFIP 8mmol/L over a time period of 6 hrs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters(protocol GE1_107_Sep09 and Grid: 028004_D_20110708) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Mar 26, 2014 |
| Last update date |
Mar 14, 2017 |
| Contact name |
Beatrice Beck-Schimmer |
| E-mail(s) |
Beatrice.BeckSchimmer@uzh.ch
|
| Phone |
0041 44 635 50 35
|
| Organization name |
University of Zurich
|
| Department |
Institute of Physiology
|
| Lab |
Group Beck-Schimmer
|
| Street address |
Winterthurerstrasse 190
|
| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE56256 |
Influence of hexafluoro-2-propanol (HFIP) on lipopolysaccharides-induced inflammation in human microvascular endothelial cells of the lung |
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