Adipocyte Differentiation from Mouse Stromal Vascular Cells

Adipose tissue from C57BL/6 mice was minced and digested with collagenase D (Roche) in a shaking water bath (37C, 225rpm, 40min). The digest was centrifuged at 400g for 10 min. Pelleted stromal vascular cells were filtered (40μm) and then washed with PBS and subjected to additional negative selection (CD31⁻/lineage⁻) adapted from previously performed methods^{44 (link)} using antibody coated microbeads (Miltenyi Biotec). Cells were cultured to confluence in collagen-coated plates and stimulated with dexamethasone, insulin and 3-isobutyl-1-methylxanthine to induce adipogenic differentiation. For genetic loss of function assays, validated shRNA sequences (Broad, Ube2e2: TRCN0000040962; Atxn1: TRCN0000240655) or scramble sequence were subcloned into a retroviral vector (pMKO.1). Gene knock-down efficiency was confirmed by qPCR in 3T3L1 cells, in each instance reproducibly achieving a minimum of 60% reduction of transcriptional activity. Differentiation into mature lipid-containing adipocytes was determined by oil-red-o (ORO) staining and quantified by measuring alcohol-extracted ORO dye at optical density 520 nm (OD₅₂₀).

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Chu A.Y., Deng X., Fisher V.A., Drong A., Zhang Y., Feitosa M.F., Liu C.T., Weeks O., Choh A.C., Duan Q., Dyer T.D., Eicher J.D., Guo X., Heard-Costa N.L., Kacprowski T., Kent JW J.r., Lange L.A., Liu X., Lohman K., Lu L., Mahajan A., O’Connell J.R., Parihar A., Peralta J.M., Smith A.V., Zhang Y., Homuth G., Kissebah A.H., Kullberg J., Laqua R., Launer L.J., Nauck M., Olivier M., Peyser P.A., Terry J.G., Wojczynski M.K., Yao J., Bielak L.F., Blangero J., Borecki I.B., Bowden D.W., Carr J.J., Czerwinski S.A., Ding J., Friedrich N., Gudnason V., Harris T.B., Ingelsson E., Johnson A.D., Kardia S.L., Langefeld C.D., Lind L., Liu Y., Mitchell B.D., Morris A.P., Mosley TH J.r., Rotter J.I., Shuldiner A.R., Towne B., Völzke H., Wallaschofski H., Wilson J.G., Allison M., Lindgren C.M., Goessling W., Cupples L.A., Steinhauser M.L, & Fox C.S. (2016). Multiethnic genome-wide meta-analysis of ectopic fat depots identifies loci associated with adipocyte development and differentiation. Nature genetics, 49(1), 125-130.

Publication 2016

collagenase D antibody coated microbeads

3 isobutyl 1 methylxanthine Adipocytes Adipogenic Adipose tissue Alcohol Antibody Assays Bath C57bl mice Cells Collagen Collagenase Dexamethasone Gene knock down Genetic function Insulin Lipid Microbeads Oil red o Optical Retroviral Shrna Stromal cells Transcriptional Vascular Vector

Corresponding Organization : Harvard University

Other organizations : Boston University, Wellcome Centre for Human Genetics, University of Oxford, Washington University in St. Louis, Wright State University, University of North Carolina at Chapel Hill, The University of Texas Rio Grande Valley, The University of Texas Health Science Center at San Antonio, UCLA Medical Center, The Lundquist Institute, Harbor–UCLA Medical Center, Universitätsmedizin Greifswald, Texas Biomedical Research Institute, University of Maryland, Baltimore, Wake Forest University, Icelandic Heart Association, Medical College of Wisconsin, Uppsala University, University Hospital of Bern, National Institutes of Health, Institute on Aging, National Institute on Aging, German Centre for Cardiovascular Research, University of Michigan–Ann Arbor, Vanderbilt University Medical Center, University of Texas Health Science Center at Dallas, Science for Life Laboratory, University of Liverpool, University of Mississippi Medical Center, Jackson Memorial Hospital, University of California, San Diego, Harvard Stem Cell Institute, Broad Institute

Top 5 similar protocols

Variable analysis

independent variables

Genetic loss of function assays using validated shRNA sequences (Ube2e2: TRCN0000040962; Atxn1: TRCN0000240655) or scramble sequence

dependent variables

Differentiation into mature lipid-containing adipocytes determined by oil-red-o (ORO) staining and quantified by measuring alcohol-extracted ORO dye at optical density 520 nm (OD520)

control variables

Adipose tissue from C57BL/6 mice
Collagenase D (Roche) digestion in a shaking water bath (37°C, 225rpm, 40min)
Centrifugation at 400g for 10 min
Filtering (40μm) and washing with PBS
Negative selection (CD31-/lineage-) using antibody coated microbeads (Miltenyi Biotec)
Culturing cells to confluence in collagen-coated plates
Stimulation with dexamethasone, insulin and 3-isobutyl-1-methylxanthine to induce adipogenic differentiation
Gene knock-down efficiency confirmed by qPCR in 3T3L1 cells, achieving a minimum of 60% reduction of transcriptional activity

controls

Positive control: Scramble shRNA sequence
Negative control: Not explicitly mentioned

Annotations

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