Anti cd206 phycoerythrin pe

Macrophages were immunolabeled with antibodies against surface proteins. The following antibodies were used: anti-F4/80 BB700, anti-CD86 allophycocyanin (APC), and anti-CD206 phycoerythrin (PE) (BD Biosciences, San Diego, USA). Macrophages were collected, and an Fc receptor blocking agent was used to block Fc receptors (Miltenyi Biotech, Germany) for 5 min at 4 °C. Macrophages were incubated with antibodies in the dark at 4 °C for 30 min and then washed with PBS. The expression of macrophage markers was calculated based on the fluorescence intensity. All of the data were collected by flow cytometry using Novo Express (ACEA NovoCyte, China) and analyzed using Flow Jo software.

RAW264.7 cells were immune-labelled with antibodies against surface proteins. The antibodies that were used were anti-CD11b fluorescein isothiocyanate (FITC), anti-CD86 allophycocyanin (APC), and anti-CD206 phycoerythrin (PE) (BD Biosciences, San Diego, USA). RAW264.7 cells were collected, and an Fc receptor-blocking agent (Miltenyi Biotech, Germany) was used to block the Fc receptors for 5 min at 4 °C. RAW 264.7 cells were incubated with the antibodies in the dark at 4 °C for 30 min and then washed in PBS. The expression of CD86 and CD206 was calculated from the fluorescence intensity. All data were collected by flow cytometry (ACEA NovoCyte, China) using Novo Express (ACEA NovoCyte, China) and analysed using FlowJo software version X (Tree Star, USA).

Stimulated RAW264.7 macrophages were removed from wells and incubated in mouse BD Fc Block (BD Biosciences, San Jose, CA) for 30 min on ice. Samples were divided and aliquots stained using anti-CD206-phycoerythrin (PE) and anti-iNOS-fluorescein isothiocyanate (FITC) (all purchased from BD Biosciences, San Jose, CA). Percentage of M1 (iNOS⁺) and M2 (CD206⁺) cells was determined by flow cytometry on a FACSCalibur (BD Biosciences, Heidelberg, Germany) and analyzed by FlowJo software (Tree Star Inc., Ashland, OR, USA).