Single-cell mass cytometry for analysis of immune system functional states

Abstract

Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with panels of up to 45 antibodies. Each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the lanthanide series of the periodic table. Antibody panels recognize surface markers to delineate cell types simultaneously with intracellular signaling molecules to measure biological functions, such as metabolism, survival, DNA damage, cell cycle and apoptosis, to provide an overall determination of the network state of an individual cell. This review will cover the basics of mass cytometry as well as outline assays developed for the platform that enhance the immunologist's analytical arsenal.

Figure 1. Mass cytometry enables high-dimensional analysis of diseases and therapies

Diseases including cancers and infections perturb cellular signaling. Mass cytometry provides a readout for up to 52 simultaneous measurements, both of those disease-induced perturbations and importantly of counter-perturbations induced by candidate therapeutics. Furthermore, the simultaneous inclusion of cell phenotype and cell cycle provide a more detailed picture than possible before. After cells are stained with antibodies and other metallic assay reagents, they are introduced into the mass cytometer as a stream of single cells, then atomized and ionized as they pass through the inductively coupled plasma (ICP) torch. Low-mass elements (including carbon and nitrogen) are filtered out by a radio frequency quadrupole before entering the time-of-flight (TOF) detector. A high-speed, online analysis system produces data equivalent to that of traditional fluorescence-based cytometry.

Figure 2. A large number of metals are available for a variety of measurements

The lanthanides provide 37 stable isotopes for measuring antigen-bound antibodies, MHC tetramers and nucleic acid probes with high sensitivity. Indium provides two additional low-sensitivity channels for highly expressed markers, and quantum dots provide one additional channel (cadmium). Rhodium and iridium, as DNA intercalators, register a cell event. Platinum, in the form of cisplatin:sulfur complexes, is used as a viability marker. Iodine, as iodo-deoxyuridine, is used for cell cycle measurements. Six palladium isotopes allow for mass tag “barcoding” and multiplexed measurement of samples.