At each endpoint the lungs were flushed with PBS supplemented with 0.5 mM EDTA (Life Technologies) three times using 1 ml for adults and 200 μl for neonates. For cAMP quantification BAL was performed using PBS. BAL cells were enumerated on counting slides (Immunesystems) using trypan blue and transferred onto microscope slides using Cytospin 4 centrifuge (ThermoFisher). Slides were stained with hematoxylin and eosin (Quick-Diff staining, Reagena). Cells were categorized as macrophages, lymphocytes/monocytes, neutrophils, and eosinophils based on morphology, coloring and size under a light microscope22 (link),58 .
Protocol detail
Bronchoalveolar Lavage Cell Analysis
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Adults
Cells
Edta
Eosin
Eosinophils
Light
Lungs
Lymphocytes
Macrophages
Microscope
Monocytes
Neonates
Neutrophils
Trypan blue
Corresponding Organization :
Other organizations : Imperial College London
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Variable analysis
independent variables
- Flushing the lungs with PBS supplemented with 0.5 mM EDTA
- CAMP quantification
- BAL cell enumeration
- Cell categorization (macrophages, lymphocytes/monocytes, neutrophils, eosinophils)
- Volume of PBS used for flushing (1 ml for adults, 200 μl for neonates)
- Staining method (hematoxylin and eosin, Quick-Diff staining)
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