Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 de-ubiquitinase activity towards c-Jun leading to its accumulation and subsequent increase in AP1-mediated gene expression [66]. A suite of highly expressed immune genes at this time supported an inflammatory type reaction in adipocytes. Hence, an unrelieved ER- stress could likely be the major cause of chronic inflam- matory responses in lipid overloaded white adipocytes in fish. Conclusions Our study revealed concordance between the gene expression profiles and the key events during adipogenic development of the primary culture of aSVF cells, including fluctuations of proliferative activity, induction of adipogenic differentiation and suppression of other mesenchymal cell lineages and final tuning of metabo- lism towards production and accumulation of lipids. For the first time in a fish species we show that the estab- lishment of mature adipocyte phenotype is associated with high activity of immune genes, activated UPR and responses to oxidative stress. These changes are likely to be part of the normal adipocyte development but may be accentuated when cells are overloaded with lipids. High expression of pro-inflammatory mediators imply that excessive growth of WAT in fish may cause dis- turbed endocrine function with possible negative effects on health, as seen in mammals. Methods Preadipocyte isolation and culture conditions Atlantic salmon was reared at Nofima Marin station at Averøy, Norway on a commercial diet to weight of 2-3 kg. Fish were anaesthetized with metacain (MS-222), bled by cut of arch bows of the gills and killed by a blow to the head. The experiment was conducted according to the National Guidelines for Animal Care and Welfare of the Norwegian Ministry of Research. Visceral adipose tissue was excised and salmon preadi- pocytes were isolated as described in Vegusdal et al. [10]. Briefly, the dissected fat tissue was washed with phosphate buffered saline (PBS, pH 7.4) (unless other- wise stated, all chemicals were obtained from Sigma- Aldrich Chemical Co., St. Louis, MO, USA), minced and digested in 0.1% collagenase (type I) in HBSS (1 g tis- sue/5 mL HBSS) at 13°C for 1 h under shaking and fil- tered through 250 and 100 μm nylon. The resulting cell suspension was then centrifuged at 700 × g for 10 min at 10°C. The buoyant fat layer with mature adipocytes was removed by aspiration, while the preadipocytes were pelleted on the bottom. After washing twice, the cells were resuspended in growth medium containing L- 15, 10% fetal bovine serum (FBS), 2 mM L glutamine, 10 mM HEPES, and antibiotics (mixture of penicillin, streptomycin and amphotericin B) and seeded on Page 13 of 17 laminin coated cell-culture flasks at a density of approximately 10 g tissue/25 cm2. The cells were kept at 13°C and media were changed every 3 days. Cells reached confluence after approximately 1 week. Conflu- ent cells were cultivated for 48 h in an differentiation inducing medium, i.e. growth medium supplemented with 1 μM dexamethasone, 33 μM biotin, 10 nM triio- dothyronine, 17 μM panthothenate and 25 μM isobutyl- methylxanthine, 20 μg/ml insulin and a lipid mixture (1 μl/ml; corresponding to 45 mg/ml cholesterol, 100 mg/ ml cod liver oil FA (methyl esters). After that cells were transferred to a maintenance differentiation media con- taining growth media only supplemented with 2 μl/ml of lipid mixture. Media was changed every 3 days until the cells reached the final differentiation stage with mor- phology of mature adipocytes (day 30). Electron microscopy Cells for electron microscopy studies were taken from cultures at days 15 and 30. Cells were washed in 0.1 M PBS (pH 7.4), then fixed in 2% glutar aldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 24 h. The cells were the harvested, rinsed in 0.1 M cacodylate buffer and post-fixed for 60 min in 2% OsO4 containing 1.5% potassium ferrocyanide, followed by en bloc staining with 1.5% uranyl acetate. Cells were dehydrated in a ser- ies of ethanol solutions (70%, 90%, 96%, and 100%) and propylene oxide, and then embedded in epon resin, which was polymerized at 60°C for 24 h. Ultrathin sec- tions (approximately 50 nm) were cut on a Reichert Ultracut E ultramicrotome using a diamond knife. The sections were placed onto formvar/carbon-coated 75- mesh copper grids, post-stained for 2 min with 0.2% lead citrate solution in 0.1 M NaOH, and examined in a Philips CM 100 transmission electron microscope at an accelerating voltage of 80 kV. Microarray analyses The samples for microarrays analyses were taken at days 1, 4, 7, 9, 12 and 30. The salmonid fish microarray (SFA2, immunochip) includes 1800 unique clones printed each in six spot replicates. Total RNA was extracted by using RNeasy® Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instruction. RNA was treated with RNase-free DNase I (Qiagen, Valencia, CA, USA), to remove any contaminating DNA. All RNA samples used in our experiments had A260/280 ratios between 1.80 and 2.30. The total RNA concentration was determined at 260 nm using spectro- photometry. Equal inputs from 5 cell flask replicates were pooled for each time point. Equalised control was made by mixing RNA from each time point. The test and control samples (15 μg RNA in each) were labelled with respectively Cy5-dUTP and Cy3-dUTP (Amersham Pharmacia, Little Chalfont, UK). The fluorescent dyes were incorporated in cDNA using the SuperScript™