Prior to euthanasia, blood was collected in heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) for subsequent single-cell analysis by flow cytometry and non-heparinized capillary tubes (Fisher Scientific) for serum collection. For cell harvests, these blood samples were then treated with ammonium-chloride-potassium lysis buffer for 5 min at room temperature and washed 1X with flow cytometry staining buffer. For serum collection, blood samples were left at room temperature for 30 min, centrifuged at 16,000 × g for 20 min, and then collected and stored at −20°C until analysis.
Bronchial alveolar lavage (BAL) fluid was collected using a protocol modified from (35 (link)). Briefly, the tracheae were cannulated with a 22-gage catheter tube (attached to a 5cc syringe) and then washed once with 1 mL of sterile PBS. Samples were stored at −20°C until analysis.
For preparation of cells from lungs and spleens, these organs were harvested after the collection of BAL fluid, digested for 30 min at 37°C in media containing 1 mg/mL Collagenase (Type 3; MP Biomedicals, Solon, OH) and 0.02 mg/mL DNase-I (MP Biomedicals), and then pressed through wire mesh to obtain a single cell suspension.
Bronchial alveolar lavage (BAL) fluid was collected using a protocol modified from (35 (link)). Briefly, the tracheae were cannulated with a 22-gage catheter tube (attached to a 5cc syringe) and then washed once with 1 mL of sterile PBS. Samples were stored at −20°C until analysis.
For preparation of cells from lungs and spleens, these organs were harvested after the collection of BAL fluid, digested for 30 min at 37°C in media containing 1 mg/mL Collagenase (Type 3; MP Biomedicals, Solon, OH) and 0.02 mg/mL DNase-I (MP Biomedicals), and then pressed through wire mesh to obtain a single cell suspension.
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