Human blood eosinophils (2×105/ml) were cultured above 0.5% w/v agarose, 48-well plate, +/− 100 pg/ml GM-CSF or GBM cell line-conditioned media generated in the presence or absence of 10 ng/ml TNF-α. In some wells, 10 µg/ml anti-GM-CSF (Clone BVD2-23B6) or isotype control antibodies were added. Cultures were maintained for 3 hr. Cells were gated (as shown in Supplementary Figure 4), negatively selected for propidium iodide stain, and positively selected for CD69 expression. (A) The percent average CD69 expression in response to media controls or conditioned media is displayed, N=5, +/− SEM, *p<0.0001 vs media, †p<0.0001 vs TNF-α, ‡p<0.0001 vs A172, §p<0.0001 vs U87-MG. (B) The percent average CD69 expression in response to co-culture with anti-GM-CSF (Clone BVD2-23B6) or isotype antibodies is displayed, N=3, +/− SEM, *p<0.0001. (C) The percent average CD69 expression in response GM-CSF cytokine depletion (Clone BVD2-23B6) or isotype controls is displayed, N=4, +/− SEM, *p<0.0001.