SVF was prepared as reported55 (link),59 (link),60 (link). Briefly, inguinal SAT or VAT were isolated from euthanized mice, minced and digested for 60 min in DMEM containing collagenase type I (2 mg/ml per gram of tissue; Invitrogen) at 37°C. Cell suspensions were then filtered through a 40-μm cell strainer and the SVF was collected as a pellet after centrifugation at 500g for 5 min. After RBC lysis, cells were stained with anti-CD45 (30-F11, BD), anti-F4/80 (clone BM8, BioLegend), anti-CD11b (M1/70, BD), anti-CD11c (HL3, BD), anti-CD206 (c06802, BioLegend; MR5D3, Acris), anti-TNF (MP6-XT22, BioLegend), anti-iNOS (CXNFT, eBioscience) and anti-CD49d [clone PS/2, AbD Serotec or clone 9C10(MFR4.B), BioLegend]. Cells were analyzed in a FACS Canto II flow cytometer (BD).
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Isolation and Characterization of Stromal Vascular Fraction
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Corresponding Organization : TU Dresden
Other organizations : Sunesis (United States), Biomedical Research Foundation of the Academy of Athens, University of Washington, Leipzig University, University of Pennsylvania, German Center for Diabetes Research, Heinrich Heine University Düsseldorf, Deutsches Diabetes-Zentrum e.V.
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Variable analysis
independent variables
- Isolation of inguinal SAT or VAT from euthanized mice
- Mincing and digestion of inguinal SAT or VAT in DMEM containing collagenase type I (2 mg/ml per gram of tissue) for 60 min at 37°C
- Expression of CD45, F4/80, CD11b, CD11c, CD206, TNF, iNOS, and CD49d in the SVF
- Cell suspension filtration through a 40-μm cell strainer
- Collection of SVF as a pellet after centrifugation at 500g for 5 min
- RBC lysis
- No positive or negative controls were explicitly mentioned in the given information.
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