Fig. 4

Glioma-secreted IL8 and CCL2 regulate GAMs to secrete TNFα a CM from NHA, or glioma cell lines was analyzed by a Proteome Profiler Human Cytokine Array and b densitometry was performed to determine the relative expression of cytokines in the CM of glioma cell lines compared to NHA. c RAW264.7 macrophages were treated with CM from NHA, U87, U251 or DMEM + 0.5% BSA containing cytokines IL-8 and/or CCL2 at concentrations of 1, 10 and 50 nM. After 24 h incubation, cells were washed once with 1x PBS and cultured in fresh DMEM + 0.5%BSA for 24 h. The supernatant was then collected and secreted TNFα was detected with TNFα ELISA kit. The relative expression of TNFα was normalized to NHA. n = 3, mean ± max/min, 95% confidence interval. *p < 0.05, **p < 0.01