Establishment of GM-IMs from murine BM.

<div><p>(A) BM cells were taken from two-month old C57BL/6 or EGFP mice. Suspensions of BM cells (3x10⁶ cells) were plated in ten cm diameter non-coated plastic culture dishes. They were cultured in RPMI-1640 supplemented with 10% GM-CSF-CM and 10% FBS. Lower panel shows phase contrast photograph of BM cultured with 10% GM-CSF-CM for six days or six months. Scale bars: 50 µm.</p> <p>(B) Growth curve of GM-IMs. BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Data shown are the mean ratios ± SE of four independent experiments.</p> <p>(C) SA-β-gal staining of BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Scale bars: 100 µm.</p> <p>(D) Expression of cellular senescent marker proteins. Cell lysates were Western blotted for p16. Β-actin was used as a control.</p> <p>(E) May-Grunwald-Giemsa staining of GM-IMs. Five months cultured GM-IMs was compared with days zero and six of BM culture in 10% GM-CSF-CM, BMMφ and peritoneal macrophages (PMφ). Scale bars: 50 µm.</p> <p>Data are representative of three independent experiments with similar results (A, C-E).</p>