To confirm the percentage of M2 macrophages in the IL-4- stimulated RAW264.7 cells, we analyzed the positive rate of CD206, a widely utilized surface marker of M2 macrophages,16 (link) by flow cytometry. Corresponding fluorescence label-conjugated isotype controls were utilized in this experiment. Briefly, all the IL-4-stimulated RAW264.7 cells were harvested 24 h later and washed by flow buffer (PBS supplemented with 1% [v/v] FBS and 0.05% NaN3) once in room temperature and once sequentially in ice-cold flow buffer. Then, CD206-specific antibody (BD Pharmingen, San Jose, CA, USA; diluted 1:40) was used to analyze the expression of CD206 via a BD Cyan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA, USA). Further analysis was performed using FloJo software (Tree Star Inc., Ashland, OR, USA).
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Quantifying M2 Macrophages in IL-4-Stimulated RAW264.7 Cells
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Corresponding Organization : Central South University
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Variable analysis
independent variables
- Stimulation with IL-4 in RAW264.7 cells
- Positive rate of CD206, a surface marker of M2 macrophages
- Incubation time (24 hours)
- Flow buffer (PBS supplemented with 1% [v/v] FBS and 0.05% NaN3)
- Fluorescence label-conjugated isotype controls
- Positive control: CD206-specific antibody
- Negative control: Fluorescence label-conjugated isotype controls
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