THP-1 macrophages were co-cultured with stably transfected CRC cells along with or without neutralizing antibodies to CCL2 (anti-CCL2; R&D Systems). Then these macrophages were processed into single cell suspensions, incubated with antibodies (BV421 Mouse anti-Human CD68, BB515 Mouse anti-Human CD86, PE Mouse anti-Human CD163, all from BD (BD Biosciences, San Jose, CA, USA) for 1 h at 4 ℃.
Mouse macrophages were then stained with CD206-APC (mouse), CD86-FITC (mouse), F4/80-PE (mouse) antibodies (eBiosciences, San Diego, CA, USA), respectively. Macrophages of nude mice subcutaneous tumor were separated and obtained using Percoll (Sigma-Aldrich, St. Louis, MO, USA) following the instruction. Flow cytometry was performed using a FACS Calibur ow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).
Mouse macrophages were then stained with CD206-APC (mouse), CD86-FITC (mouse), F4/80-PE (mouse) antibodies (eBiosciences, San Diego, CA, USA), respectively. Macrophages of nude mice subcutaneous tumor were separated and obtained using Percoll (Sigma-Aldrich, St. Louis, MO, USA) following the instruction. Flow cytometry was performed using a FACS Calibur ow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).