Ifn γ c746

Primary AMs were isolated by repeatedly flushing mouse lungs with 1 ml of PBS eight times at room temperature to obtain BAL. Cells were centrifuged at 1,500 rpm for 5 min, washed twice using DMEM complete medium, and then cells were incubated for 3 hours in 24-well plates at a density of 3 x 10⁵ cells/well to allow cells to adhere. Next, the cells were washed twice with PBS to remove non-adherent cells for subsequent processing. To generating peritoneal macrophages, intraperitoneal injections of 3% thioglycolate broth were administered, and then cells were extracted and harvested for assays. To generate BMDMs, bone marrow cells were cultured in macrophage complete medium containing 20 ng/ml M-CSF (CB34, Novoprotein). After 6 days in culture, non-adherent cells were eliminated and adherent cells were harvested for assays. Next, the AMs, PMs and BMDMs were cultured in 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml IFN-γ (C746, Novoprotein) for M1 polarization, or 20 ng/mL interleukin-4 (CK15, Novoprotein) for M2 polarization treatment for up to 24 hours. M1 and M2 macrophages were harvested for quantitative PCR and western blotting analysis.

17β-Estradiol (50-28-2), β-glucan (G-59303) from yeast were purchased from XiEnSi biotechnology company (Tianjin, China). DPI was purchased from Selleck (Shanghai, China). M-CSF (#CB34) and IFN-γ (#C746) for BMDMs were purchased from Novoprotein (Shanghai, China). LPS was purchased from Sigma (St. Louis, MO, USA). Antibody of NOX2 (19013-1-AP), Beclin1 (66665-1-Ig) and Rubicon (21444-1-AP) were purchased form proteintech (Wuhan, China). Estrogen receptor β (ab288) was purchased from Abcam (Shanghai, China). P-Akt (Ser235/236, #4060), Akt (#4691), Estrogen receptor α (13258s), β-actin (#3700), LC3B (#83506), ATG5 (#12994) and ATG7 (#8558) were purchased from Cell Signaling (Boston, MO, USA).