Stromal Vascular Fraction Analysis

Stromal vascular fraction (SVF) was isolated as described previously (23 (link), 24 (link)). Briefly, sAT and gAT were isolated and immediately minced and digested with type I collagenase (Gibco, Darmstadt, Germany). Digested AT was filtered through 100 μm cell strainer (BD, Heidelberg, Germany), and the adipocyte fraction was removed by centrifugation. SVF cells in the pellet were washed and resuspended in FACS buffer (PBS, 0.1%BSA, 0.1%NaN₃) and analyzed for the content of inflammatory and immune cells by using flow cytometry (FACS Canto II, BD Bioscience). Mφs were detected as CD11b⁺ F4/80⁺; M1 Mφs were detected as CD11b⁺ F4/80⁺ CD11c⁺ cells. Total T cells (CD3⁺) were further divided into T helper (CD3⁺ CD4⁺) and cytotoxic T cells (CD3⁺ CD8⁺). Fc receptors on leukocytes were blocked by anti-CD16/CD32 antibody (BD, Heidelberg, Germany). The following fluorescently labeled antibodies were used: CD11b-APC, CD11c-PE (BD, Heidelberg, Germany), F4/80-Alexa-fluor 488 (eBioscience, Frankfurt, Germany), CD8a-APC, CD4-FITC, CD3e-PE (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).