Abstract
Classical four-color fluorescence flow cytometry helped define the major cell subsets of the immune system that we understand today (i.e. T-cells, B-cells, macrophages). Machines with eight or more colors brought characterization of rare immune subsets and stem cells. With intracellular staining, higher parameter measurements lead to examination of regulatory signaling networks and patient stratification with clinical outcomes. However, this progression has now been stymied by the limits of fluorescence spectral overlap considerations in fluorophore-based tagging methods. Now, mass cytometry (CyTOF) offers examination of 30–50 parameters without fluorescent agents or interference from spectral overlap. Essentially, heavy metal isotopes as reporters replace fluorophores. By then exploiting the resolution, sensitivity, and dynamic range of elemental mass spectrometry, on a time-scale that allows the measurement of 1000 individual cells per second, this device offers a much-simplified alternative for ultra-high content cytometric analysis. Using mass cytometry (Science 2011) combined with novel single-cell visualization methods (SPADE Nature Biotechnology 2011) we examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of most cell subsets spanning a hematopoietic hierarchy defined by this profiling was monitored with 18 simultaneous functional markers and a battery of ex vivo stimuli and inhibitors. We will also provide examples of how high dimensional mass cytometry analysis can reveal previously unappreciated levels of organization in virus-specific memory T cell compartments (Immunity 2012), and massively multiplexed single-cells kinase inhibitor profiles on primary samples (Nature Biotechnology 2012). Together, these collective works expose unappreciated layers of human hematopoietic organization, and provide an opportunity to reevaluate diseases and pharmacological therapeutics as specific perturbations to this inherent order.