|
| Status |
Public on Jul 17, 2007 |
| Title |
Intraabdominal isolated adipocytes, rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Isolated adipocytes from intraabdominal adipose issue
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Gender: male
Age: 42 to 49 days
Tissue: epididymal adipose tissue
|
| Growth protocol |
To obtain purified cell fractions, ten 6- to 7-week-old C57BL/6 males were killed, and epididymal and flank subcutaneous adipose tissue was removed under sterile conditions. Tissues from each depot were pooled, minced, and digested with 1 mg/ml collagenase for 45 min at 37°C in DMEM/Ham’s F-12 1:1 (DMEM/F12) containing 1% BSA and antibiotics (100 units/ml penicillin, 0.1 mg/ml streptomycin, 2.5 mg/ml fungizone, and 50 mg/ml gentamicin). Digested tissues were filtered through sterile 150-µm nylon mesh and centrifuged at 250 × g for 5 min. The floating fraction consisting of pure isolated adipocytes was then removed and washed two more times before proceeding to RNA extraction. The pellet, representing the SVF containing preadipocytes and other cell types, was resuspended in erythrocyte lysis buffer consisting of 154 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA for 10 min. The cell suspension was centrifuged at 500 × g for 5 min and then resuspended in a culture medium consisting of DMEM/F12, 10% FCS, and antibiotics. This cell suspension was filtered through a 25-µm sterile nylon mesh before being plated on a 10-cm plate at 60,000 cells per cm². Sixteen hours after plating, cells were extensively washed with PBS and then subjected to RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy extraction of total RNA was performed according to the manufacturer's instructions (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
Double-stranded cDNA synthesis was reverse-transcribed from 15 µg of isolated mRNA by using the SuperScript Choice system (Invitrogen) with an oligo(dT) primer containing a T7 RNA polymerase promoter site. Double-stranded cDNA was purified with Phase Lock Gel (Eppendorf). Biotin-labeled cRNA was transcribed by using a BioArray RNA transcript labeling kit (Enzo).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 µg of cRNA were hybridized for 16 hr at 45C on GeneChip Murine Genome U74Av2 . GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 G7
|
| Description |
Isolated adipocytes from intraabdominal adipose issue
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
|
| |
|
| Submission date |
Jul 17, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Stephane Gesta |
| E-mail(s) |
[email protected]
|
| Organization name |
Joslin Diabetes Center
|
| Street address |
One Joslin Place
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL81 |
| Series (1) |
| GSE8505 |
Isolated adipocytes and stromo-vascular fraction (SVF) of subcutaneous and intraabdominal adipose tissue in mice |
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