Pharmingen staining buffer

After different treatments, RAW264.7 cells and BMDM cells were resuspended in BD Pharmingen staining buffer (cat# 554657; BD Biosciences). Cells were incubated with FC block (cat# 553141; BD Biosciences) for 20 minutes. Subsequently, cells were washed and resuspended in 100 μl of BD Pharmingen staining buffer and incubated with PE Rat Anti-Mouse F4/80 (cat# 565410; BD Biosciences), FITC Rat Anti-CD11b (cat# 557396; BD Biosciences), PE-Cy^TM7 Rat Anti-Mouse CD86 (cat# 560582; BD Biosciences), Alexa Flour® 647 Rat Anti-Mouse CD206 (cat# 565250; BD Biosciences) on ice for 30 minutes. Cells were washed three times with staining buffer and resuspended in staining buffer. Cells were centrifuged and resuspended in staining buffer for FACS analysis (FACS Celesta; BD Bioscience, San Jose, USA). FACS data analysis was performed using FLOWJO^TM Software.

Harvested cells were washed and fixed in cold ethanol (70%) for 2 h at −20°C. Then, cells were washed twice using PBS and once in a BD Pharmingen staining buffer (BD Biosciences, 554,656). Resuspension of cell pellets was done in BD Pharmingen PI/RNase staining buffer (0.5 ml; BD Biosciences, 550,825) followed by 15 min of incubation at room temperature (RT). Detection of red fluorescence was done at an excitation wavelength of 488 nm, after which the abundance of cells at G0/G1, S, as well as G2/M phases were determined by the ModFit software.