Gene Expression & Cell Subsets Figure 1. Matrix of scatter plots showing estimated log2-fold changes for each cell population. The log-fold changes for each sample are plotted against those for each of the other samples for gene expression after stimulation with LPS for 3 hours (top right of diagonal) and 24 hours (bottom left). Correlation coefficients between each pair of samples are shown in the bottom right of each box. doi:10.1371/journal.pone.0004427.g001 Effects on individual gene expression The expression of a number of specific individual genes involved in the immune response to LPS was investigated further to compare expression in different cell types. First, the expression of ‘validation’ genes encoding cytokines likely to be differentially expressed in monocytes in response to LPS on the basis of previous published data was investigat- ed[8,9,10]. The expression of interleukin (IL)-1a, IL-1b, IL-6 and IL-10 genes was upregulated as expected, with expression after 3 hours’ LPS stimulation being more upregulated than after 24 hours (figure 5). There was concordance in expression for these differentially expressed genes in monocytes and PBMC samples. TNF expression was increased at 3 hours as expected and, although statistically significant, did not quite reach the cut-off for fold change. This likely reflects the fact that TNF expression induced by LPS is known to peak earlier than 3 hours[11]. Likewise, there were other inflammatory response genes differentially expressed after LPS stimulation that were detected in both PBMC and monocytes. These included genes that were upregulated, such as monocyte chemotactic protein 2 (MCP2), and genes that were downregulated, such as dendritic cell immunoreceptor (DICR), confirming that genes strongly differen- tially expressed in monocytes can be detected in PBMC (figure 6). Of particular interest were genes that had significant differential expression after LPS stimulation in monocytes that were not detected when PBMC were used for analysis (figure 7). The linker for activation of T cells (LAT) gene, known to be upregulated in monocytes[12], was significantly upregulated at 3 hours in the Mono+ and Mono2 samples. Conversely, expression of this gene was significantly downregulated in the MonoD sample, resulting in an overall lack of change in expression in the PBMC sample. There were, however, also genes where the differential expression in response to LPS appeared markedly different between monocytes and PBMC, but were detectable in both samples. For example, the small inducible cytokine A5 (RANTES)[13] was highly differentially expressed in the Mono+ sample. Although PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4427