Cytometry A. sections, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL?1) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that this prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR 1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort. = 10) or preterm (= 9) birth (Panel 1). Within 30 min of venipuncture, individual whole blood aliquots were stimulated with different concentrations of LPS (0, 0.1, 1, 10, and 100 ng mL?1), fixed, and frozen at Fanapanel ?80C (Panel 2). For each LPS concentration, all samples were barcoded using a combination of three palladium (Pd) mass tags, pooled, and processed simultaneously (Panel 3). Pooled samples were stained using a combination of 27 cell-surface markers and 11 functional markers (Panel 4) and analyzed by mass cytometry (Panel 5). The resulting dataset was normalized to account for changes in machine sensitivity and then de-barcoded (Panel 6). Unsupervised hierarchical clustering and manual gating strategies Fanapanel were applied to visualize and quantify patient-specific signaling responses in immune cell subsets spanning the entire immune system. Shown is a visual representation of a cluster hierarchy plot (Panel 7). Contoured are clusters that fall within canonical immune cell subsets. Immune features (cell frequency or signaling responses) that differed significantly between the term and preterm study groups were identified using two complementary statistical approaches (Panel 8). Assaying whole blood General considerations The assay was performed in whole blood samples kept at room temperature rather than WAGR in PBMCs to minimize sample processing actions and preserve immune cells in as close to in vivo conditions as possible. Importantly, samples were stimulated with external ligands (if applicable), fixed, and stored at ?80C within 60 min of whole blood collection. There are several important differences between assaying whole blood or PBMCs. Cells in whole blood are fixed within 60 min of collection, while PBMCs are frozen in liquid nitrogen as live cells. Because cells in whole blood are fixed before being stored, stimulation of these cells with external ligands has to occur before storage. In contrast, PBMCs are stimulated after samples are removed from storage and thawed. However, fixing and storing immune cells directly in whole blood samples has the advantage of preserving all immune cell populations (including granulocytes) and avoiding a density gradient centrifugation step common in PBMC preparations, which may alter immune cell distribution, cell-surface antigen expression, transcriptional activity (15C19), and introduce potential elemental contaminants (e.g. iodine, barium Fanapanel and other) (20). Stimulation with external ligands to evoke cellular responses General considerations Stimulation of whole blood samples with external ligands occurs within 30 min of sample collection. The choice of ligand(s) is based on the biological question under investigation. In essence, ligands are chosen to perturb signaling pathways in cell subsets that are implicated in disease-related and pathophysiologically important processes in order to unmask disease-specific cellular alterations that may not be detectable in non-perturbed cells (21). Typically, supra-physiological ligand concentrations are used to evoke the maximum response, thereby testing a cells functional capacity (1,21). However, stimulation with physiologically more relevant concentrations may reveal biologically important differences in cellular responses that are impartial of their functional capacity. The importance of mimicking physiological conditions was highlighted in a recent article by Kay et al. demonstrating that polyfunctionality in natural killer (NK) and T cells to the pH1N1 virus was increased during pregnancy, while responses to the non-physiological ligands phorbol 12-myristate 13-acetate and ionomycin were reduced (22). In the current study, the exploration of ligand concentration versus response functions allowed for a more comprehensive Fanapanel characterization of cellular functions. Specific protocol In this study, LPS was chosen as it selectively binds to the toll-like receptor 4 (TLR4). TLR4 signaling plays an important role in the maintenance of pregnancy (23,24). More specifically, in mice, intrauterine infusion of LPS reproducibly induces preterm birth, a phenomenon that depends on the presence of a functional TLR4.