Facs 3

After establishing direct co-culture for 24 h, RS504393 (CCR2 inhibitor) and Cetuximab (EGFR inhibitor) were added into the co-culture system. Control group was performed in the same way and received the same medium but without medicine. After 24 h, the co-culture system was sorting by BD FACS III according to green fluorescence and size of cells. For flow cytometric analysis, the sorted THP1 cells were harvested and suspended in ice-cold PBS containing 3% BSA (Sigma). 5 × 10⁵ cells/tubes in 100 μl were prepared and incubated for 30 min. Cells were washed and incubated with PE anti-human CD163 and Alexa Fluor^® 647 anti-human CD206, as well as corresponding isotype antibodies for 30 min in dark. After washed 3 times, cells were analyzed on FACS Calibur flow cytometer (BD Biosciences) using Cell Quest software.

Cells were collected and rinsed in cold PBS before being incubated with 5 μl Annexin V-FITC and 5 μl PI (Vazyme, China) at 4°C for 10 mins without light. The percentage of apoptosis cells was assessed using a BD FACS III flow cytometer (BD, USA).