Apoptosis was detected by staining cells with Annexin V-FITC/propidium iodide (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. CD14Ab (Biolegend, CA, USA) staining was used to exclude Mφs from the analysis.
Expression of active caspase-3 was measured using a Fluorescein Active Caspase-3 Staining Kit (Invitrogen, Carlsbad, CA, USA). Expression of CD206, CD163, and CD86 was measured by direct immunofluorescence using PE-conjugated CD206 (Biolegend, CA, USA) and CD163 (Biolegend, CA, USA) and APC-conjugated CD86 (Biolegend, CA, USA). An isotype control was used to exclude nonspecific signals.
For intracellular CCL2 staining, cells were stimulated with Leukocyte Activation Cocktail (BD Bioscience, CA, USA) for 4 h at 37 °C. The cells were then fixed and permeabilized with Fixation Buffer and Intracellular Staining Perm Wash Buffer (Biolegend, CA, USA) according to the manufacturer’s instructions. Next, the cells were stained with a PE-mouse anti-human MCP-1 antibody (BD Pharmingen, San Diego, CA, USA). Data were acquired with a FACScan flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo 7.6.1.
Expression of active caspase-3 was measured using a Fluorescein Active Caspase-3 Staining Kit (Invitrogen, Carlsbad, CA, USA). Expression of CD206, CD163, and CD86 was measured by direct immunofluorescence using PE-conjugated CD206 (Biolegend, CA, USA) and CD163 (Biolegend, CA, USA) and APC-conjugated CD86 (Biolegend, CA, USA). An isotype control was used to exclude nonspecific signals.
For intracellular CCL2 staining, cells were stimulated with Leukocyte Activation Cocktail (BD Bioscience, CA, USA) for 4 h at 37 °C. The cells were then fixed and permeabilized with Fixation Buffer and Intracellular Staining Perm Wash Buffer (Biolegend, CA, USA) according to the manufacturer’s instructions. Next, the cells were stained with a PE-mouse anti-human MCP-1 antibody (BD Pharmingen, San Diego, CA, USA). Data were acquired with a FACScan flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo 7.6.1.