Flow cytometric analysis
After the spleen was taken out, rinsed in RPMI-1640 complete medium,then lysed the red blood cell and adjusted the cell concentration to 1×106/ml with PBS and stained with the following antibodies: F4/80-PE-CY7(Biolegend), CD11b-FITC(BD PharmingenTM), CD206-BV650(Biolegend), Arg-1-APC(R&D), iNOS-PE(eBiosience), and CD40-BV421(BD Pharmingen TM). For detection of extracellular molecules, the cells were incubated for 30 min at 4°C with antibodies(F4/80, CD11b, CD206, Arg-1, CD40). For detection of intracellular molecules(iNOS), cells were permeabilized with the Fixation/Permeabilization Solution Kit(BD PharmingenTM) for 20 min, then incubated with the antibodies for 30 min at 4°C. In FACS analysis, both F4/80 and CD11b were positive to calculate the proportion of macrophages(Q2). In macrophages, iNOS or CD40 positive was identified as M1 macrophages(Q1+Q2+Q3), and CD206 or Arg-1 positive was identified as M2 macrophages(Q5+Q6+Q7)[7].