How CyTOF works - the technology
Mass cytometry combines high sensitivity of ICP-TOF technology with flow cytometry for proteomic analysis at the single cell level
Mass cytometry is a revolutionary technology invented by Scott Tanner (University of Toronto and DVS Sciences - acquired by Fluidigm in 2014 (Bandura DR et al., 2009, doi: 10.1021/ac901049w). Mass cytometry is a hybrid technology that combines flow cytometry and mass spectrometry and is also known as cytometry by time-of-flight (CyTOF).
CyTOF technology is based on the concept of using heavy-metal isotopes to label antibodies rather than fluorescent tags. These antibodies are then used to label cells and the labeled cells are introduced into a mass spectrometer for quantitative detection of the metal labels associated with each cell (Figure 1). The use of metal tags allows the combination of many more antibody specificities in a single experiment, without significant spillover between detector channels. BRFAA has a 3rd generation CyTOF instrument (Helios) that allows for the analysis of panels of up to 40 antibodies, along with live/dead markers, barcoding reagents, and a DNA intercalator to identify intact cells.
Data produced can be exported as FCS files and can be analyzed using conventional flow cytometry software (like Flowjo from Treestar).
However, the complexity of the polyparametric multidimensional CyTOF data requires alternative analytical techniques (algorithms) such as viSNE, SPADE, Citrus, Phenograph, Cen-se (ref) and others which can be used in cloud-based analysis platform like Cytobank (link) as well the pathsetter software (ref).
Figure 1. Mass cytometry allows single-cell atomic mass spectrometry of heavy elemental (> 100 Da) reporters. Schematic of ICP-MS-based analysis of cellular markers. An affinity product (e.g., antibody) tagged with a specific element binds to the cellular epitope (1 and 2). The cell is introduced into the ICP by droplet nebulization (3). Each cell is atomized, ionized (4 and 5), overly abundant ions removed (5 and 6), and the elemental composition of remaining heavy elements (reporters) is determined (7). Signals corresponding to each elemental tag are then correlated with the presence of the respective marker and analyzed using conventional cytometry platforms (8) (taken and modified from Bendall S. et al., 2012, doi: 10.1016/j.it.2012.02010)
Mass cytometry Workflow
Familiriase with the mass cytometry workflow to obtain quality data and save effort, time and money
The 5 Basic elements of mass cytometry
It's about metals, panels, resolution, channels and millions of cells.