|
| Status |
Public on Oct 31, 2012 |
| Title |
Indirect +PMN 2 |
| Sample type |
RNA |
| |
|
| Source name |
T84, 2hrs post-conditioned media, from migration with PMN, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: T84
cell type: intestinal epithelial
migration model: Indirect
neutrophils: yes
|
| Treatment protocol |
On day of experiment monolayers were transferred to Hanks Buffered Saline, with 1uM fMLP on the apical surface (bottom chamber), to establish chemotactic gradient. Neutrophils were freshly isolated from healthy volunteer venous blood and used for either direct or indirect migration.
|
| Growth protocol |
T84 intestinal epithelia were cultured in DMEM/F12 with 10% FCS at 37oC with 5% CO2. Experimental cells were seeded and grown to confluence (TER>1,000 Ohms.cm2) on the underside of 3cm2 3um-pore Transwell permeable supports (Corning).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using TRIzol reagent (Invitrogen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >7.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression 2hrs after incubation with cell-free supernatants from cells after neutrophil transmigration
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Signal intensities were normalized using combined rank consistency filtering with LOWESS intensity normalization in Agilent Feature Extraction software. Data was subsequently analyzed by GeneSpring (Agilent).
|
| |
|
| Submission date |
Jul 26, 2012 |
| Last update date |
Oct 31, 2012 |
| Contact name |
Eric L Campbell |
| E-mail(s) |
eric.campbell@ucdenver.edu
|
| Organization name |
University of Colorado - Anschutz Medical Campus
|
| Street address |
12700 East 19th Ave
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE39681 |
Transcriptional imprinting of transmigrating neutrophils on T84 intestinal epithelial cells |
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