Glogiplug

Fleshly isolated cells were rested in PBS supplemented with 0.5–1% FBS for 2h. Then, they were stimulated with varying doses of recombinant murine GM-CSF (Peprotech), recombinant murine M-CSF (Peprotech) or vehicle (PBS) for 30 min. In some experiments, cells were pretreated by rapamycin (Sigma-Aldrich) as indicated. For an in vitro culture assay, cells harvested by bronchoalveolar lavage were labeled with CFSE or CellTrace where indicated. Cells were cultured in complete RPMI-1640 medium containing 10% FBS, 2 mM glutamine, and penicillin and streptomycin (100 U/ml each; Invitrogen) for 1–2 h at 37°C and 5% CO₂. The non-adherent cells were discarded, and the plates were washed by warm PBS twice. The remaining adherent cells were used as alveolar macrophages, and were cultured in the presence of 10 ng/ML LPS (Sigma-Aldrich), 10 ng/ML recombinant murine IL-4 (Peprotech), 10 ng/ML recombinant murine GM-CSF, recombinant murine M-CSF or vehicle (PBS) for the indicated times. The cultured cells constituted more than 95% as alveolar macrophages verified by flow cytometry analysis. GlogiPlug (BD Bioscience) was added if intracellular flow cytometry analysis required. 0.5 μM 4-OHT (4-hydroxy-tamoxifen; Sigma-Aldrich) were administered into the culture medium for the induction of Cre-mediated deletion.