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| Status |
Public on Jun 27, 2007 |
| Title |
1. IMR differentiated 3D T84 culture |
| Sample type |
RNA |
| |
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| Source name |
Human intestinal epithelial T84 cells (CCL 2´48, ATCC Rockville, MD, USA)
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| Organism |
Homo sapiens |
| Characteristics |
T84 cells are induced to differentiate by the soluble factors secreted by the IMR fibroblasts. The epithelial crypt type cells are differentiated to enterocyte s.
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| Biomaterial provider |
ATCC
|
| Treatment protocol |
Human intestinal epithelial T84 cells (CCL 2´48, ATCC Rockville, MD, USA) were cultured in three-dimensional type I collagen gel as previously described Halttunen et al. 1996. T84 cells were induced to differentiate by soluble factors secreted by IMR-90 type human embryonic lung fibroblasts (CCL 186, ATCC). IMR fibroblasts, cultured on the top of the epithelial cells, were separated from the epithelial cells by cell-free collagen layer. The cells were cultured 7 days in incubator.
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| Growth protocol |
Human intestinal epithelial T84 cells (CCL 2´48, ATCC Rockville, MD, USA) were cultured in three-dimensional type I collagen gel as previously described Halttunen et al. 1996. T84 cells were induced to differentiate by adding 20 ng/ml human recombinant TGFbeta1 (hTGF-beta1, R&D Systems Europe, Oxon, UK). The cells were cultured 7 days in incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mRNA was extracted from the cell culture samples (only from T84 cells) to ice-cold TRIzol reagent (Life Technologies, Inc. Frederick, MD, USA) according to the manufacturer�s protocol. All samples were subjected to DNAse I treatment (Roche Diagnostics GmbH, Mannheim, Germany). Total RNA was quantitated by spectrophotometry and quality checked by agarose gel electrophoresis.
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| Label |
33P dCTP (ICN Radiochemicals)
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| Label protocol |
Probe preparation and microarray hybridization were performed following manufacturer's (Research Genetics) protocol using 1.5 ug total RNA as template, 10 ul (10mCi/ml) 33P dCTP (ICN Radiochemicals), 1,5 ul dNTP mix containing dATP, dTTP, dGTP at 20 mM (Pharmacia ), 1,5 ul reverse transcriptase (Supercript II, Life Technologies), 1,0 ul DTT. Elongation for 90 min at 37oC. The label was purified with Bio-Spin 6 Chromatography column (Bio-Rad),
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| Hybridization protocol |
Array was hybridized for 12 h at 42oC in roller bottle. After hybridization membrane was washed twice in 2 X SSC 1%SDS, 0.5XSSC.
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| Scan protocol |
The hybridized membrane was let to expose Phosphoimager screens for 12 h. Hybridization signals were detected on a Storm 860 phosphoimager (Molecular Dynamics, Amersham Biosciences, Buckinghamshire, England)
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| Description |
The normalisation was done in excell with sum method (Kroll et al. 2002)
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| Data processing |
Filter images were aligned and spot intensities analysed with Pathways Software (Research Genetics). Raw data were imported to Microsoft Excel.
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| Submission date |
Jun 27, 2006 |
| Last update date |
Jun 27, 2006 |
| Contact name |
Kati Marjaana Juuti-Uusitalo |
| E-mail(s) |
lokaju@uta.fi
|
| Phone |
+358-335518404
|
| Fax |
+358-335518402
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| Organization name |
Pediatric Research Center
|
| Department |
Medical School
|
| Street address |
Biokatu 10
|
| City |
Tampere |
| ZIP/Postal code |
33014 |
| Country |
Finland |
| |
|
| Platform ID |
GPL3928 |
| Series (1) |
| GSE5170 |
TGFbeta- and IMR induced epithelial cell differentiation in a three-dimensional intestinal crypt-villus axis model |
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