Lung tissue was digested as described above to obtain a single-cell suspension. SiglecF+ cells were isolated using anti-SiglecF microbeads (Miltenyi Biotec) and plated in non-treated 6-well plates in RPMI 1640 media containing 1x GlutaMAX, 1x pyruvate, 1x penicillin/streptomycin, 10% fetal bovine serum (FBS), and 20 ng/ml recombinant GM-CSF (Biolegend), as previously described (31 (link)). Culture medium was replaced after 16 h of incubation at 37°C and again every 2 days in culture. Cells were collected on days 1 or 6 after plating. Where indicated, AMs were treated overnight with 5 μM of the Wnt/β-catenin inhibitor XAV939 (Sigma) or 1 μM of the Stat3 inhibitor Napabucasin (Fisher Scientific) before cells were harvested.
Protocol detail
Isolation and Culture of Alveolar Macrophages
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Cells
Culture medium
Fetal bovine serum
Gm csf
Lung
Microbeads
Napabucasin
Penicillin
Pyruvate
Stat3
Streptomycin
Tissue
Xav939
β catenin
Corresponding Organization :
Other organizations : Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute, National Institutes of Health, National Cancer Institute, University of California, San Diego
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Variable analysis
independent variables
- Treatment with Wnt/β-catenin inhibitor XAV939 (5 μM)
- Treatment with Stat3 inhibitor Napabucasin (1 μM)
- Characteristics and behavior of SiglecF+ cells on days 1 and 6 after plating
- Single-cell suspension obtained from lung tissue
- SiglecF+ cells isolated using anti-SiglecF microbeads
- Cells plated in non-treated 6-well plates in RPMI 1640 media containing 1x GlutaMAX, 1x pyruvate, 1x penicillin/streptomycin, 10% fetal bovine serum (FBS), and 20 ng/ml recombinant GM-CSF
- Culture medium replaced after 16 h of incubation at 37°C and again every 2 days in culture
- Positive control: Not specified
- Negative control: Untreated SiglecF+ cells
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