Fcs express software

The retinal cells were stained with fluorescent dyes conjugated to antibodies as follows: CD206-FITC (C068C2; BioLegend, San Diego, CA, USA); 7-AAD (BD Pharmingen, San Jose, CA, USA); CD11b-APC-Cy7 (M1/70; BD Biosciences San Jose, CA, USA); f4/80-PE-Cy7 (BM8; BioLegend). After staining, the cells were washed twice with a FACS buffer (0.5% BSA in PBS buffer) and suspended in the FACS buffer for FACS analyses. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software, Los Angeles, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages.
To investigate intracellular cytokine production, retinal cells were treated with a leukocyte activation cocktail with BD GolgiPlug^TM (BD Biosciences) for 4.5 h and with a BD Cytofix/Cytoperm Fixation/Permeabilization^TM kit (BD Biosciences) and then stained with the following antibodies: TNF-α-FITC (MP6-XT22; eBiosciences, San Diego, CA, USA); IL-10-PE (GK1.5; BioLegend); CD11b-APC-Cy7 (M1/70; BD Biosciences); f4/80-PE-Cy7 (BM8; BioLegend); and 7-AAD (BD Pharmingen, San Jose, CA, USA). The 7-AAD− CD11b+ f4/80+ cells were defined as retinal macrophages. Data were collected using a FACS Canto II flow cytometer (BD Biosciences) and analyzed by FCS Express software (De Novo Software).

Cells were isolated from AMC and Coleman fat at the indicated time points. Briefly, the fat was digested (30 minutes on a shaker at 37° C) in PBS containing 0.075% collagenase. Mature adipocytes and connective tissue were removed by centrifugation at 800 × g for 5 minutes. The cell pellets were resuspended and filtered through a 100 μm mesh and a 70 μm mesh. Total cells were then counted. Then, the cells were stimulated for 4 h with 20 ng/ml phorbol myristate acetate (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) prior to addition of 10 μg/ml brefeldin A (BFA) (eBiosciences). For the detection of surface markers, cells were stained with CD31 (eBiosciences), CD34 (eBiosciences), CD90 (eBiosciences), and CD45 (eBiosciences) and then incubated for 15 min at 4° C in the dark. After washing, cells were fixed and permeabilized using fixation buffer and permeabilization buffer (BD Biosciences). Acquisition was performed on a Coulter Epics-XL flow cytometer using the System II software (Coulter Corporation, Brea, CA, USA). Data analysis was performed using FCS express software (De Novo Software, Los Angeles, CA, USA).