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ECM950
Sigma-AldrichAdipogenesis Assay
The Adipogenesis Assay provides a convenient format for induction & analysis of adipogenesis in the classic 3T3-L1 model.
More>>The Adipogenesis Assay provides a convenient format for induction & analysis of adipogenesis in the classic 3T3-L1 model. Less<<
Adipogenesis Assay: SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.
Obesity is a significant clinical problem that contributes to life-threatening diseases such as diabetes and atherosclerosis. The process of adipogenesis, the formation of adipose tissue, has become better understood by the study of several cell types that can be induced to undergo differentiation into adipocytes. The first, and best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line (Kehinde and Green, 1974). 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets (Figure 1; Rubin et al., 1978). Another cell line, 3T3-F442A, is blocked from differentiating at a later stage, and requires only insulin to differentiate.
The biochemical pathways of adipogenesis have become increasingly well understood with the use of the 3T3-L1 model (reviewed in Gregoire et al., 1998; Rosen et al., 2000). Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer-binding protein b (C/EBPb). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels (Elks and Manganiello, 1985). At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBPd. C/EBPb and d in turn induce transcription of C/EBPa and PPARγ. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation (Tang et al., 2003). Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation (Nakae et al., 2003). C/EBPa and PPARγ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin.
Endogenous negative regulators of adipocyte differentiation, such as Pref-1 and Wnt-10b, are highly expressed on undifferentiated 3T3-L1 cells, and are down-regulated upon addition of adipogenesis inducers (Ross et al., 2000; Mei et al., 2002). In addition, cytokines such as tumor necrosis factor alpha (TNFa) and transforming growth factor beta (TGF-b) interfere with adipocyte differentiation (Gregoire et al., 1998).
The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. PPARγ has received particular attention as a target, as it is essential for the final phase of adipocyte differentiation and is a pharmacological target for the thiazolidinedione class of antidiabetic drugs. The pro-adipogenic effect of the thiazolidinediones has led to interest in identifying compounds that retain antidiabetic activity without promoting adipogenesis. In addition, inhibitors of PPARγ activity have been identified that inhibit adipogenesis, and might serve as the basis for development of effective anti-obesity drugs (Wright et al., 2000; Camp et al., 2001).
Materials Required but Not Delivered
· 3T3-L1 Preadipocyte cell line (can be obtained from ATCC: ATCC# CL-173).
· Media for propagation of 3T3-L1 cells - DMEM with 10% calf serum (not fetal calf serum) and antibiotics.
· Adipogenesis basal media - DMEM with 10% fetal calf serum and antibiotics.
· 24-well or other size tissue culture plates for induction of adipogenesis.
· Spectrophotometer or 96-well plate reader capable of detecting 490 nm or 520 nm.
References
Product Information
Components
IBMX Solution - (Catalog No. 90355) - One vial containing 250 μl of 0.5 M 3-isobutyl-1-methylxanthine (IBMX) in DMSO.
Insulin Solution - (Catalog No. 90356) - One vial containing 250 μl of 10 mg/mL recombinant human insulin.
Dexamethasone Solution - (Catalog No. 90357) - One vial containing 100 μl of 10 mM dexamethasone in ethanol.
Oil Red O Solution - (Catalog No. 90358) - One bottle containing 60 mL 0.36% Oil Red O solution in 60% isopropanol.
Wash Solution - (Catalog No. 90360) - Two bottles containing 250 mL each of wash solution.
Dye Extraction Solution - (Catalog No. 90359) - One bottle containing 30 mL of dye extraction solution.
The Adipogenesis Assay provides a convenient format for induction & analysis of adipogenesis in the classic 3T3-L1 model.
Application Notes
The Chemicon Adipogenesis Assay provides a convenient format for induction and analysis of adipogenesis in the classic 3T3-L1 model. The most commonly used inducers of adipogenesis, dexamethasone, IBMX and insulin, are provided in convenient, ready-to-use formulations. In addition, reagents for staining of differentiated adipocytes are provided to allow the investigator to obtain quantitative information about inducers and inhibitors of adipogenesis. A solution of Oil Red O is used to stain lipid droplets in mature adipocytes. A wash solution is provided to remove free Oil Red O from the cell layer. Also included is Dye Extraction Solution that extracts lipid-bound Oil Red O for measurement in a spectrophotometer (Figure 2).
Biological Information
Physicochemical Information
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Materials Information
Toxicological Information
Safety Information according to GHS
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Product Usage Statements
Usage Statement
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Storage and Shipping Information
Storage Conditions
Note: Kit components require two different storage temperatures.
Dexamethasone Solution, IBMX Solution and Insulin Solution should be stored at -20ºC. Oil Red O Solution, Wash Solution, and Dye Extraction Solution should be stored at room temperature. Storage of Oil Red O Solution and Dye Extraction Solution at -20ºC may result in formation of insoluble precipitates and is not recommended. If Oil Red O solution forms a precipitate, remove particulates by passage through a 0.22 or 0.45 micron filter.· Oil Red O stains skin and clothing. IBMX and dexamethasone are irritants and potentially toxic. DMSO is readily absorbed through the skin. Wear a lab coat and gloves when handling these solutions.
Precautions: Isopropanol is flammable. Keep solutions containing isopropanol (Oil Red O Solution, Wash Solution and Dye Extraction Solution) away from open flames.
Gene expression profile of dental pulp cells during differentiation into an adipocyte lineage. Tadashige Nozaki,Kiyoshi Ohura Journal of pharmacological sciences
115
2010
Gene regulation during in vitro differentiation into adipocytes was examined in rat dental pulp-derived cells. Insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were added to induce adipogenesis. Cells containing lipid droplets were observed after induction as in 3T3 L1 cells. Rat dental pulp-derived cells showed their potential to differentiate into adipocytes in vitro. In both types of cells, the pluripotent markers Oct-3/4 and Sox2 were downregulated during differentiation, whereas the expression of Nanog was not significantly changed during differentiation. Interestingly, in the dental pulp-derived cells, the level of Oct-3/4 was transiently induced at 1 week after induction and then significantly decreased during differentiation. Based on the expression profiles determined using GeneChip Arrays, 3418 probes across 10 clusters showed a difference in expression at 1, 2, and 3 weeks after induction versus before induction. Notably, genes in the PPAR signaling pathway including Ppar?, Fabp4, and the C/EBP family were upregulated by more than 3-fold. Upregulation of the PPAR pathways seems to be a critical signal transduction pathway in this differentiation system. These findings indicate that dental pulp-derived cells are a potential source of adipogenic cells, and their gene expression profile could be useful in future regenerative medicine applications.
