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Status |
Public on Apr 24, 2015 |
Title |
HSC_sample_1 |
Sample type |
RNA |
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Source name |
HSC cells from patient 1
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Organism |
Homo sapiens |
Characteristics |
cell type: Primary human hepatic stellate cells (HSCs)
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Treatment protocol |
For stimulation of HCC cells with HSC conditioned media, HCC cells were seeded into T25 flask (10^6 cells). One day after seeding, cells were washed with serum-free DMEM, and then incubated for another 12 h with serum-free DMEM. Subsequently, the medium was changed and cells were incubated with 3 mL of HSC-CM or control medium (serum-free DMEM) for 4h.
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Growth protocol |
The human HCC cell lines Hep3B (American Type Culture Collection (ATCC) number HB-8064) was cultured as described (Amann et al, 2009, Cancer Science). Primary human hepatic stellate cells (HSCs) were isolated from 15 different human donors as described in (Amann et al, 2009, Cancer Science). The isolation procedure and cell culture on uncoated tissue culture dishes led to the activation of HSCs. For collection of conditioned medium (CM) HSCs were seeded into T75 flasks (2 × 10^6 cells). One day after seeding cells were washed twice with serum-free DMEM, and then incubated for another 24 h with serum-free DMEM (15 mL/T75). CM was clarified by centrifugation at 6,000 g to remove cell debris, sterile filtered (0.45 μm pore size membrane filter), and stored in aliquots at −80 °C until use. Serum-free DMEM incubated for 24 h in cell culture flasks without cells served as the control.
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Extracted molecule |
total RNA |
Extraction protocol |
Isolation of total cellular RNA from cultured cells and tissues and reverse transcription were performed as described in Amann et al., 2009, Cancer Science.
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Label |
biotin
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Label protocol |
The Ambion WT Expression Kit Protocol was used for the preparation of biotinylated sense-strand cDNA from 300ng of total RNA, followed by fragmentation and labeling according to the Affymetrix GeneChip WT Terminal Labeling and Hybridization User Manual.
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Hybridization protocol |
3.8 μg of fragmented and labeled sense-strand cDNA were hybridized for 16 h at 45°C on GeneChip Human Gene 1.0 ST microarrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
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Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
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Description |
Gene expression of hepatic stellate cells (HSC)
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Data processing |
Raw intensity values from CEL files were normalized with the Bioconductor package VSN. Probes were summarized using median polish and an alternative CDF based on ensembl genes provided by BrainArray, University of Michigan (version 12.1.0).
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Submission date |
Oct 17, 2014 |
Last update date |
Apr 25, 2015 |
Contact name |
Julia C Engelmann |
E-mail(s) |
julia.engelmann@ur.de
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Organization name |
University of Regensburg
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Department |
Statistical Bioinformatics
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Street address |
Josef-Engert-Str. 9
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City |
Regensburg |
ZIP/Postal code |
93053 |
Country |
Germany |
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Platform ID |
GPL19306 |
Series (1) |
GSE62455 |
Gene expression of paired samples of hepatic stellate cells (HSC) and hepatocyte cell culture (HCC) treated with conditioned media of HSC cells |
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