Gene Expression & Cell Subsets Figure 6. Genes expressed in common between monocytes and PBMC. Examples of gene expression that is (a) upregulated and (b) downregulated in both PBMC and monocyte samples after stimulation with LPS for 3 and 24 hours. The log2-fold change (M value) and standard error for the expression of each gene are plotted for each sample. doi:10.1371/journal.pone.0004427.g006 undertaken by means of titration experiments[16]. A series of artificially mixed samples could be used, with, for example, decreasing proportions of MonoD added to Mono+ samples. We chose to study the interaction between monocytes and LPS because these inflammatory cells comprise a significant minority of PBMC (in this study 10–14%) and their interaction with LPS in initiating the inflammatory cascade in Gram negative bacterial infection is well described. It is likely that our results apply to the detection of gene expression in other situations. The proportion of genes in a particular cell type that can be detected in a cell mixture might be lower for target cells comprising a smaller proportion of the mixture or in which the cell type of interest is not the principal target or primary effector cell. However, it is probable that detection of the most differentially expressed genes would remain less susceptible to the influence of other cells within the cell mixture. To ensure gene expression induced by LPS stimulation was comparable in the monocytes analysed alone and in those analysed within PBMC, monocytes were separated after stimulation of PBMC with LPS, thus preserving the interactions between cells. The repeatability of our results was confirmed by the high degree of correlation between the two PBMC samples analysed from the same individual. This control validated the relevance of the differences in gene expression detected between PBMC and the monocyte samples. We included a number of other control samples to exclude the possibility that our results were confounded by artefactual or technical variability. Monocytes were isolated by different methods to enable us to control for the possibility that gene expression might be induced by binding of CD14 antibodies during positive selection of monocytes. The similarity in differential gene expression between the Mono+ and Mono2 samples showed that changes in cell surface expression of CD14 or LPS-binding of CD14 did not have a major effect. The greater correlation between the PBMC and Mono+ samples than between the PBMC and Mono2 samples reflects the higher proportion of monocytes selected using positive isolation. Negative isolation resulted in up to 50% of non-monocytes cells remaining in the Mono2 sample. Possible explanations include the relatively lower efficacy of negative selection and the absence of antibodies to every non- monocyte cell type present in PBMC. Artefactual or technical variability as a cause of differences in gene expression between samples was also excluded by the finding that the samples that shared the most similar technical processes (Mono+ and MonoD) had the most different gene expression, whilst those that underwent the most different processes (Mono+ and Mono2) had the most similar expression. Our study has important implications for the design and analysis of microarray-based studies. For studies in which detection of subtle changes in gene expression in a specific cell type is important, our study shows that analysing a cell mixture will miss a substantial proportion of changes. However, when only the most highly differentially expressed genes are of interest, it may not be necessary to undertake time-consuming and costly separation and analysis of individual cell types. Materials and Methods This study was reviewed and approved by the Human Research Ethics Committee (23096A) at the Royal Children’s Hospital, and informed consent was obtained verbally from the adult volunteers. PBMC separation and stimulation Blood from two healthy volunteers was collected into tubes containing endotoxin-free lithium heparin (Becton Dickinson, Franklin Lakes, NJ, USA). PBMC were separated using a Ficoll- PaqueTM gradient (Amersham Biosciences, Uppsala, Sweden). Triplicate aliquots from each individual of 26106 PBMC were simultaneously stimulated with 1 mg/ml LPS (Sigma Aldrich, Sydney, NSW, Australia) and incubated at 37uC with 5% CO2 for 0, 3 and 24 hours. The triplicates at each time point were treated identically to enable comparison between the PBMC samples and two monocyte samples that would subsequently be separated. TRIzolH (Invitrogen Life Technologies, Carlsbad, CA, USA) was added to all samples before storing at 280uC. Monocyte separation Monocytes were separated by two different methods. Positive isolation: monocytes were positively selected by incubation for 60 min at 4uC with magnetic DynabeadsH M-450 CD14 (Dynal PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4427