Peritoneal macrophage and stromal vascular (SV) cells of epididymal adipose tissues were fractionated as described[18 (link),19 (link)]. Briefly, to get peritoneal macrophage, 5 ml of cold phosphate buffer saline (PBS) was injected into mouse peritoneal cavities immediately after anesthesia. After shaking the mice for 2-3 min, peritoneal fluid was harvested and spun down for peritoneal macrophages at 500 g for 5 min at 4 °C. The stromal vascular cells were isolated from the equal mass of epididymal adipose tissues using the collagenase digestion method. For flow cytometry analysis, same quantity cells (1 × 106) were subsequently re-suspended and stained with appropriate antibodies (F4/80 and CD11c for M1 type macrophage, or F4/80 and CD206 for M2 type macrophage) as described in our previous study[20 (link)]. Antibody information used in flow cytometry analysis is as follows: 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). All data were collected using FACScan and analyzed using CellQuest software (BD Biosciences, San Jose, CA).
Fitc anti mouse cd11c antigen
Manufactured by BD
Sourced in United States
FITC anti-mouse CD11c antigen is a fluorescently-labeled monoclonal antibody that binds to the CD11c surface marker expressed on mouse dendritic cells. This product is intended for use in flow cytometry and other immunoassays to identify and quantify CD11c-positive cells.
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3 protocols using fitc anti mouse cd11c antigen
1
Isolation and Characterization of Murine Peritoneal Macrophages and Adipose Stromal Cells
2
Isolation and Characterization of Mouse Peritoneal Macrophages
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PM were isolated as described previously [41 (link)]. Briefly, about 5 mL of cold phosphate buffer saline (PBS) was injected into mouse abdominal cavities. After vigorously shaking the mice for 2 min, solution in abdominal cavity (the PBS containing PM) was carefully collected. PM was obtained by centrifugation at 1000 g for 5 min. For flow cytometry analysis, equal amounts of the PM cells (1 × 106 in 100 μL PBS) were incubated with appropriate antibodies; PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA, USA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA, USA), and APC anti-mouse CD206 antigen (BD Bioscience). The flow cytometry data were collected using a FACScan and analyzed with Cell Quest software (BD Biosciences). Macrophages labeled with F4/80+CD11c+CD206− were counted as pro-inflammatory M1-like macrophages and those labeled with F4/80+CD11C−CD206+ were counted as anti-inflammatory M2-like macrophages.
3
Isolation and Characterization of Adipose SVF
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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).
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