Isolation and Characterization of Murine Adipose-Derived Stromal Cells

Murine ASC were obtained from 6–8 weeks old C57Bl/6J mice (Charles River Laboratories). The isolation of stromal-vascular fraction from adipose tissue was carried out as previously described^{27 (link),38 (link)}. Briefly, extracellular matrix was digested with Collagenase A (Roche) and centrifuged at 1200 g. The pellet obtained was re-suspended and the stromal fraction was collected by sequential centrifugation and filtration steps. Cells were then cultured in complete medium (DMEM + glucose + GlutaMAX ITM + 15% heat-inactivated adult bovine serum + penicillin/streptomycin; all from Invitrogen) supplemented with 50 ng/ml HB-EGF (R&D Systems). The immune phenotype of murine ASC was characterized by using monoclonal antibodies (mAb) specific for CD106, CD9, CD44, CD80, CD138, and Stem cells antigen 1 (Sca1). In addition, the absence of hematopoietic (CD45, CD11c, and CD34) and endothelial markers (CD31) was assessed as previously described^{6 (link)}. All mAbs were purchased from BD Pharmingen (San Diego). Isotype-matched antibodies were used as controls. For immunophenotypic analysis, ASC were incubated at 4 °C for 10 min with 15% adult bovine serum followed by incubation with the specific mAb at 4 °C for 30 min. At least 10,000 events were analyzed by flow cytometry on a FACScalibur (Becton Dickinson) using the Cell Quest software (Becton Dickinson).