Alexa Fluor® 647 Rat Anti-Mouse CD206

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Mouse lung alveolar macrophages were collected by fitting a venous indwelling needle into a mouse trachea. PBS was injected to wash the lung six consecutive times (0.8 ml/wash). Total bronchial alveolar lavages were collected and washed with PBS.	In brief, cells were washed once in room temperature flow buffer (PBS supplemented with 1% (v/v) FBS and 0.05% NaN3) and once in ice‐cold flow buffer, and cell staining was performed on ice. Samples were analysed using 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).

Upstream tips
Mouse lung alveolar macrophages were collected by fitting a venous indwelling needle into a mouse trachea. PBS was injected to wash the lung six consecutive times (0.8 ml/wash). Total bronchial alveolar lavages were collected and washed with PBS.
Protocol tips
In brief, cells were washed once in room temperature flow buffer (PBS supplemented with 1% (v/v) FBS and 0.05% NaN3) and once in ice‐cold flow buffer, and cell staining was performed on ice. Samples were analysed using a BD Cyan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA, USA).
Downstream tips
). Further analysis was performed using FloJo software (Tree Star Inc., Ashland, OR, USA).

Publication protocol

Mouse lung alveolar macrophages were collected by fitting a venous indwelling needle into a mouse trachea. PBS was injected to wash the lung six consecutive times (0.8 ml/wash). Total bronchial alveolar lavages were collected and washed with PBS. The expression of surface markers was analysed using Abs specific for mouse CD206, CD86 and CD68 (all purchased from BD Pharmingen, San Jose, CA, USA). Corresponding fluorescence label‐conjugated isotype controls were utilized in all experiments. In brief, cells were washed once in room temperature flow buffer (PBS supplemented with 1% (v/v) FBS and 0.05% NaN3) and once in ice‐cold flow buffer, and cell staining was performed on ice. Samples were analysed using 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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