Experimental design and sample processing for microarray analysis of GBM-induced molecules in co-cultured myeloid cells.

<p>(<b>a</b>) ABI1700 cDNA microarrays were used to analyze global changes in gene transcripts using a cutoff in the change of gene expression of > 2 fold. We analyzed the expression profile of 29,362 human genes using RNA samples from unstimulated monocytes (n=8), LPS-stimulated monocytes (n=8), LPS-stimulated monocytes cocultured with glioblastoma cells (n=8), and monocytes cocultured with human astrocytes (n=2). Of these, 14,414 gene expression profiles were used for subsequent analysis, as this set of genes passed all quality control criteria. Local Pooled Error Tests (PLPE package from Bioconductor) were performed comparing gene expression values of monocytes stimulated with LPS in the absence vs. presence of GBM cells and subsequently stimulated monocytes co-cultured with GBM cells vs. stimulated monocytes co-cultured with primary human astrocytes. (<b>b</b>) After 4 hours of culture in 4 experimental conditions, monocytes were isolated from GBM or human astrocytes based on FSC/SSC properties. Purity was re-confirmed in a small fraction of each sample based on CD11b expression.