|
| Status |
Public on Dec 30, 2012 |
| Title |
ATM CD34(-) rep1 |
| Sample type |
RNA |
| |
|
| Source name |
human adipose tissue resident macropgages, CD34(-)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: adipose tissue resident macropgages, CD34(-)
|
| Treatment protocol |
CD34-positive AT resident macrophages (ATMs), CD34-negative ATMs and adipose stromal cells were isorted from aspirated AT by a multiparameter fluorescent-activated cell sorter. Circulating monocytes were also sorted from peripheral blood.
|
| Growth protocol |
Liposuction aspirates were obtained from healthy donors undergoing liposuction of the abdomen and thighs.the aspirated fat was washed with phosphate-buffered saline (PBS) and digested on a shaker at 37℃ in PBS containing 0.075% collagenase for 30 minutes. Mature adipocytes and connective tissue were separated from pellets by centrifugation. Peripheral blood was obtained from healthy volunteers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were isolated from each samples using ISOGEN (Nippon Gene) according to the manufactureres’ recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled using the ‘Agilent Quick-Amp Labeling Kit’ (Agilent Technologies) following the manufacturer’s protocol. Cy3-labeled cRNA was purified with the RNeasy kit (Qiagen). Dye incorporation was assessed with the NanoDrop-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.4 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4851A) 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), 1 minute with 37°C GE Wash buffer 2 (Agilent), and 30 second at room temperature with acetonitrile, then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides..
|
| Description |
Gene expression of CD34(-) ATMs
|
| 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_F_20101102 ) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non positive and significant, and Feature Non above background outliers were excluded.
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| |
|
| Submission date |
Apr 30, 2012 |
| Last update date |
Dec 30, 2012 |
| Contact name |
Hisako Ishimine |
| Organization name |
National Institute of Advanced Industrial Science and Technology
|
| Department |
Research Center for Stem Cell Engineering
|
| Street address |
higashi1-1-1 Tsukuba central 4
|
| City |
Tsukuba |
| State/province |
Japan Ibaraki |
| ZIP/Postal code |
305-8562 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE37660 |
Transcription profile of adipose-resident macrophages and mesenchymal stem cells. |
|
