Fitc conjugated anti mouse cd86 antibody

The cell surfaces were stained with a phycoerythrin (PE)-conjugated anti-mouse F4/80 antibody (Biolegend 123110, California, United States), APC-conjugated anti-mouse CD206 (MMR) antibody (Biolegend 141708, California, United States), and FITC-conjugated anti-mouse CD86 antibody (Biolegend 105006, California, United States). The expression of F4/80 was determined using flow cytometry to identify the macrophages. The expression of CD86 was used to delineate the M1 macrophages, while the cells stained by the APC-conjugated anti-mouse CD206 (MMR) antibody could be identified as M2 macrophages. The cell suspensions were stained for 30 min on ice with specific antibodies and washed two times with 3 ml of PBS buffer supplemented with 0.5% bovine serum albumin (BSA). Finally, we analyzed it using flow cytometry (CytoFlex A00-1-1102, California, United States).

HUVEC were treated with or without AGEs (100 μg/ml), ARF (1%) or melatonin (20 μM), as mentioned for the in vitro apoptosis assay. Approximately 1 × 10⁶ cells were analyzed by the fluorescent dye annexin V-FITC/propidium iodide (PI) to detect apoptotic cells (annexin V+/PI− and annexin V+/PI+).
RAW264.7 cells were cultured to 40% confluence in DMEM containing AGEs (100 μg/ml), and treated with or without ARF (1%) and melatonin (20 μM). Moreover, certain cells were stimulated with LPS (100 ng/ml, M1 polarized) or IL-4 (40 ng/ml, M2 polarized) for activation as the positive control. Following digestion and collection, cells were stained with 1% BSA containing the FITC-conjugated anti-mouse CD86 antibody (Biolegend, 159204, USA) or PE-conjugated anti-mouse CD206 antibody (Biolegend, 141703, USA) to confirm the polarization of macrophages, respectively. All data were analyzed using a BD Accuri C6 flow cytometer.