Fig 2: M2 macrophages protect human glioma from chemotherapeutic agent cisplatin (CDDP)-induced apoptosis through the generation of nitric oxide (NO) in vitro. (A) Immunofluorescence staining of human glioma samples with anti-CD206 antibody (green) and anti-iNOS antibody (red). DAPI (blue) was used for nuclei detection (scale bar = 20 µm). Panels on the right represent enlarged image details. (B) U373 cells were treated with increasing concentrations of CDDP for 24 h before the MTT assay. Data are expressed by setting the absorbance of the reduced MTT in the absence of CDDP as 100%. The data points represent the results obtained from four independent experiments. (C) Flow cytometry analysis of U373 cells proliferation at 24 h after treatment with CDDP (35 µg/ml) or untreated (UT). The data are represented as fold change of proliferation index compared with UT (n = 3). **p < 0.001 vs UT. (D) Inducible nitric oxide synthase (iNOS) expression in human M2 macrophages, assessed by western blotting. N9 cells treated with LPS were used as positive control. GAPDH was used as the internal standard. The images are representative of three independent experiments. (E) Evaluation of CDDP-induced apoptosis of U373 cells cultured alone or in the presence of M2. l-NAME (2 mM) and DETA-NO (20 µM) were added 1 h before CDDP (50 µg/ml, 24 h) administration. Panel on the right shows apoptosis quantification expressed as fold increase of total apoptotic cells (annexin V+/PI- and annexin V+/PI+ cells) compared to their respective UT controls (dashed line) (n = 4). **p < 0.001, ***p < 0.0001 vs UT; ++p < 0.001, +++p < 0.0001 vs CDDP; ##p < 0.001 vs CDDP + M2; §p < 0.05 vs l-NAME + CDDP + M2.

Fig 3: Surface receptor expression and phagocytic activity of differentiated cells. G‐LPS‐MΦs (A, C) and M‐IL‐4‐MΦs (B, D) were fixed with 5% formalin, then blocked and stained with anti‐MHC class II (A, B) or anti‐CD206 (C, D) antibodies. Merged images of fluorescence and counterstaining with DAPI show the expression of MHC class II (A; white arrows) on G‐LPS‐MΦs and CD206 (D) on M‐IL‐4‐MΦs. Uptake of fluorescent latex beads in G‐LPS‐MΦs (E) and M‐IL‐4‐MΦs (F). The cells were incubated with fluorescently labeled opsonized latex beads for 90 min. After washing with phosphate buffer, cells were stained with trypan blue. The fluorescent image is overlaid on phase contrast image of cells. Bars represent 50 μm. The total fluorescence collectively derived from the internalized beads and cell surface‐associated particles was quantified by measuring the fluorescent intensity using a fluorescence spectrometer (G). This assay was performed in quintuplicate and repeated two times. Statistical analysis was performed using the Student's t‐test. *P ≤ 0.00001.