Lung DC migration assays using PKH as a fluorescent tag were done as described previously.41 (link) To analyze costimulatory molecules on lung DCs, mouse airways were instilled with OVA plus LPS, and the animals euthanized 16 h post-treatment with an overdose of pentobarbital sodium. Following perfusion of the pulmonary vasculature with PBS, lungs were excised and placed in ice cold PBS containing 0.5% BSA. DCs from minced and digested lung tissue were isolated as previously described70 and stained with antibodies against I-Ab and CD11c. For analysis of co-stimulatory marker expression, DCs were evaluated by flow cytometry using antibodies against the following molecules: I-Ab (AFb.120) CD103 (M290), CD11c (N418), CD11b (M1/70), CD40 (Ic10), CD80 (16-10A1), and CD86 (GL1), BD Biosciences, BioLegend and eBioscience, San Diego, CA.
Protocol detail
Lung DC Costimulatory Molecule Analysis
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Animals
Antibodies
Cd103
Cd11b
Cold
Flow cytometry
Lung
Migration assays
Mouse
Overdose
Pentobarbital sodium
Perfusion
Tissue
Corresponding Organization :
Other organizations : National Institutes of Health
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Variable analysis
independent variables
- Instillation of OVA plus LPS into mouse airways
- Expression of co-stimulatory markers (CD40, CD80, CD86) on lung dendritic cells (DCs)
- Expression of cell surface markers (I-Ab, CD103, CD11c, CD11b) on lung DCs
- Time point of analysis (16 h post-treatment)
- Perfusion of pulmonary vasculature with PBS
- Isolation of lung DCs from minced and digested lung tissue
- Negative control: Not explicitly mentioned
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