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Stromal vascular fraction was isolated from epididymal fat as described previously [26 (link),27 (link)]. Briefly, about 1 g fresh epididymal fat was weighted and cut into small pieces with scissors in digestion buffer (Krebs-Ringer bicarbonate buffer (KRB) supplemented with 1 mg/mL collagenase Type I (Worthington Chemicals, Lakewood, NJ, USA)). After incubation for 30 min in 37 °C shaking water bath, the tissue slurry was filtered through nylon mesh to remove undigested tissue. After centrifuged at 2200 rpm, the pellet of stromal vascular fraction (SVF) was re-suspended and washed with KRB twice. Then, about 1 × 10⁶ SVF cells were re-suspended in PBS (100 μL) and incubated with corresponding antibodies. The antibodies of PE anti-mouse F4/80 (eBioscience, San Diego, CA, USA), FITC anti-mouse CD11c (BD Bioscience, San Jose, CA, USA) and purified CD16/CD32 (BD Bioscience) were used to label macrophages. The remaining SVF cells were incubated with nonspecific IgG (BD Bioscience) to evaluate background fluorescence. The flow cytometry analysis was carried out on a FACScan (BD Biosciences).

Stromal vascular fraction was isolated as described previously [65 (link), 66 (link)]. Briefly, 1g of epididymal adipose tissue was dissected and minced in Krebs-Ringer bicarbonate buffer (KRB) containing 1 mg/ml collagenase Type I (Worthington Chemicals, Lakewood, NJ). The solution was incubated in 37°C water bath for 30 minutes. The tissue slurry was then filtered through nylon mesh to remove undigested tissue, and centrifuged at 2200 rpm to fractionate adipocytes and stromal vascular fraction (SVF). SVF cells (1 × 10⁶ in a volume of 100 μl of PBS) were incubated with antibodies for flow cytometry analysis. The antibodies included PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA), purified CD16/CD32 antigen (BD Bioscience, San Jose, CA), and APC anti-mouse CD206 antigen (BD Bioscience, San Jose, CA). Cells were incubated with nonspecific IgG to assess background fluorescence (BD Bioscience, San Jose, CA). The data were collected using a FACScan and analyzed using CellQuest software (BD Biosciences, San Jose, CA).