Cytometry by Time-Of-Flight (CyTOF)


PI/Head: Olaf Rotzschke, Ph.D.

CyTOF Services
Manager: Karen Teng
Email: Karen_Teng@immunol.a-star.edu.sg 

The SIgN CyTOF facility houses a next-generation flow cytometer that can acquire >40 independent parameters per cell allowing deep immunophenotyping of the immune response

Background
CyTOF, or mass cytometry, uses molecularly tagged antibodies to detect and quantify specific cellular antigens, allowing for highly multiplexed assays. The SIgN CyTOF facility houses a next-generation flow cytometer that employs a type of mass spectrometry approach known as Inductively Coupled Plasma – Mass Spectrometry (ICP-MS). Here, heavy-metal isotopic tags rather than fluorophores are used to tag antibodies (or peptide-MHC tetramers in this case) and stain the cells. This method of isotopic tagging incurs less crosstalk between channels than fluorophore-based tagging, and an increasing number of channels (>40) are available for detection. Once tagged, the cells are sprayed into a plasma torch to break the chemical bonds; here, the cellular contents ionizes and the elemental content of all atoms with a molecular weight between 100 and 200 atomic mass units is quantified.  The SIgN mass cytometer can discriminate between differences in atomic mass at the level of a single atomic mass unit (Figure 1)


Figure 1: Mass cytometry approach

Technologies and Approach
The CyTOF® instrument was originally developed by Tanner et al., in Toronto, Canada (Figure 2). This instrument can acquire >40 independent parameters per cell and uses custom antibody panels that are tailored to specific cellular analyses. Novel multiplexing technology in combination with this system is under development at the SIgN CyTOF facility so that a large number of T-cell antigen specificities can be probed within the same sample.1 Using this approach, the phenotypic and functional characteristics of these cells can be evaluated in depth.2



Figure 2: CyTOF® systems installed at SIgN

Multi-parameter analysis of single cells by flow cytometry provides a wealth of information about individual cells.  The information content for each cell increases exponentially with the number of parameters acquired. Therefore, the amount of information that can be gained from a single cell using CyTOF and >40 cellular parameters is vast. For example, using a simple means of analyzing cells, each cell can be classified as positive or negative for each parameter. Using 10-color flow cytometry leads to 210 = 1,024 possible colour combinations for each cell; 40-parameter CyTOF analysis leads to 240 > 1 trillion possible metal combinations for each cell. There are many methods to analyse the CyTOF data output, including SPADE clustering analysis,3 Principal Component Analysis and Boolean gating (Figure 3).2


Figure 3: High-dimensional cellular analysis. (a) SPADE clustering analysis. This example plot shows CD8+ T cells from a healthy donor and each node is colored by relative CD45RA expression. (b) Principal component analysis. High-dimensional data is compressed into composite dimensions, each of which is composed of the weighted sum of all original parameters. (c) Boolean gating. The 512-pixel heat plot summarizes the relative frequencies of all possible combinations of nine different functional capacities of CD8+ T cells.1-3

References
1.Newell, E.W., et al. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat Methods. 6(7) 497-499. 2009
2.Newell, E.W., et al. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity. 36 (1): 142-152, 2012.
3.Bendall, S.C., et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 332 (6030): 687-696, 2011.