Cell culture and treatments

Human malignant glioma cell lines U251, A172 and human monocytic cell line THP-1 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (CBTCCCAS; Shanghai, China). The cell lines had been recently authenticated based on DNA fingerprinting, isozyme detection and cross species checks. All cells were maintained in suspension in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 95% air, 5% CO2.

To generate THP-1-derived M2-polarized macrophages (M2 cells), THP-1 cells (in a six-well plate, 1 × 10⁶ cells/well) were treated with 100 ng/ml PMA for 6 h, and then cultured with PMA plus 20 ng/ml IL-4 and 20 ng/ml IL-13 for another 18 h. For production of the conditioned medium from M2 cells (M2-CM), all PMA, IL-4 and IL-13 were removed by a thorough wash, and M2 cells were further cultured in 2 ml fresh serum-free DMEM medium for another 24 h. In order to prepare conditioned medium from THP-1 cells (THP-1-CM), THP-1 cells were cultured in 2 ml fresh medium for 24 h. Then the conditioned medium were collected respectively and stored at −80°C.

Human peripheral blood monocytes (PBMs) from healthy donors were isolated by density-gradient centrifugation using Ficoll-Hypaque (Pharmacia, Peapack). Monocytes were isolated using anti-CD14 microbeads (Miltenyi Biotec), and more than 97% of the cells acquired were CD14⁺ monocytes as determined by FACS analysis (data not shown). For in-vitro monocyte-derived M2 macrophages activation, CD14⁺ monocytes at 1 × 10⁶ cells/mL were treated for 5 days with 100 ng/ml recombinant human macrophage colony-stimulating factor, followed by 20 ng/ml IL-4 and 20 ng/ml IL-13 for 3 days. This study was approved by the institutional review board of Qilu Hospital of Shandong University. Informed consent was obtained from each subject.