Gene Expression & Cell Subsets Figure 7. Genes expressed differentially between monocytes and PBMC. Examples of gene expression that is (a) upregulated in monocytes but not PBMC, (b) upregulated in monocytes and remains detectable in PBMC and (c) downregulated in monocytes but not PBMC after stimulation with LPS for 3 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.g007 Biotech, Carlton South, Victoria, Australia) coated with anti- CD14 monoclonal antibody. The positively-selected monocytes were isolated from the sample with a Dynal MPCH magnet and the monocyte-depleted supernatant transferred to another tube and centrifuged. Negative isolation: monocytes were isolated by the removal of other cell types using a commercial kit (DynalH Monocyte Negative Isolation Kit). In addition to gamma globulin to block Fc receptors on monocytes, a mixture of mouse monoclonal antibodies for CD2, CD7, CD16, CD19, CD56 and CD 235a was added to the PBMC sample. After incubating and washing the cells, the Depletion DynabeadsH coated with an Fc specific human antibody against mouse IgG were added and incubated for 30 min at 4uC. A Dynal MPCH magnet was used to isolate beads attached to the unwanted cells. The supernatant containing the negatively-isolated monocytes was removed and centrifuged. After isolation, TRIzolH was added to the three samples: positively-isolated monocytes (Mono+), the cellular fraction depleted of monocytes after positive isolation (MonoD) and the negatively-selected monocytes (Mono2), before storing at 280uC. Cell population analysis by flow cytometry For each of the PBMC, Mono2 and MonoD samples, 56105 cells were stained with PE-conjugated CD14 (IOTestH, Immuno- tech, Marseille, France) and 56105 cells were stained with PE- conjugated mouse IgG1 as a negative isotype control. Cells were PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4427