Mass Cytometry in Single-Cell Analysis

Mass cytometry™ is a variation of flow cytometry that measures cellular properties using metal ion-conjugated antibodies in lieu of fluorescently tagged ones. The technology complements a growing suite of single-cell technologies, each of which has their own advantages, disadvantages, and applications. Compared to technologies like flow cytometry and single-cell transcriptomics, mass cytometry offers a useful compromise in terms of the depth of information obtained, accessibility, resolution, and, in some cases, cost.

Metals versus fluorophores

Mass cytometry and flow cytometry are similar in that they measure the properties of cells in suspension. But they differ in how they measure these properties. Whereas conventional flow cytometry measures fluorescence, mass cytometry detects lanthanide metal isotopes.

Mass cytometry’s use of metal signals instead of fluorescent ones obviates concerns over signal overlap and autofluorescence that arise in flow cytometry experiments, allowing the analysis of up to 50 markers on millions of cells in a single experiment.¹ These markers can include surface and intracellular markers as well as cell signaling events (via phosphorylated proteins, for example).

Metal-labeled antibodies also simplify panel design. “Because CyTOF technology uses mono-isotopic metals, panel design is very simple and straightforward,” says Andrew Quong, Chief Science Officer at Fluidigm. “The only caveats are spillover due to purity and oxidation of the metal tags which need to be accounted for, but these effects are relatively small.”

Another advantage of labeling cellular features with metals instead of fluorophores is stability. Not only are mass cytometry samples always fixed, they’re also not photosensitive like flow samples. Both of these characteristics make it possible to ship samples elsewhere when there’s no local expertise or capability.

Timothy Bushnell, Associate Professor and Scientific Director of the University of Rochester (URMC) Flow Cytometry Resource, has been the recipient of such samples and can attest to their stability. “We had a sample that came from across the country. It got left on the tarmac — during the summer. When we finally got it and ran it, the control sample looked identical to the control from the previous experiment,” says Bushnell.

While the cost of metal-tagged and fluorescently tagged antibodies is similar, comparing the cost of mass cytometry and flow cytometry experiments is difficult because each technology has different requirements. “Cost comes down to what the experiment is. In some cases, we found that mass cytometry can end up being slightly cheaper,” says Matthew Cochran, Technical Director of the URMC Flow Cytometry Resource. “With flow cytometry, you often end up having to run more controls.”

To make mass cytometry more affordable and user-friendly, Fluidigm, the only vendor of mass cytometry reagents and equipment, launched a new system in 2021: the CyTOF XT. “The innovation embedded in this new system simplifies operation, increases throughput, integrates new sample introduction automation, improves time to results, and reduces the total cost of ownership,” says Thiru Selvanantham, Senior Product Manager at Fluidigm.

Proteins versus transcripts

Mass cytometry was originally developed to expand the number of targets analyzable by cytometry, making it ideal for immunophenotyping. Yet, while single-cell transcriptomic technologies can be used to define cellular populations and states within the immune system, there are a number of reasons a lab might choose mass cytometry instead.

“Transcriptomic techniques can look at many more markers, but at the end of the day, proteins are your main effector molecules,” says Herman Li, a laboratory technician in Handy Gelbard’s lab at URMC. In collaboration with Niccolo Terrando’s lab at Duke, the Gelbard lab is leveraging mass cytometry to understand how inflammation leads to delirium after orthopedic surgery.

“We’re biased to begin with. In our mouse model of delirium, we’re programmed to think microglia are the key players. But the thought we might miss a temporally limited population of other immune cells that might be more pivotal is what attracted us to mass cytometry,” says Gelbard. “With single-cell transcriptomics, you’re frequently confronted with too much information and sometimes hypotheses get obfuscated by that.” Mass cytometry is also substantially cheaper and less bioinformatically intensive than single-cell transcriptomics.

The two labs’ focus on immune cells in the brain has led them to consider imaging mass cytometry (IMC), a variation of mass cytometry that “makes use of metal label antibodies and detection probes rather than fluorescently labeled probes to image slides” instead of cells in suspension, according to Quong.

Adding the spatial dimension to mass cytometry can make the technology more approachable, particularly for those who prefer to work with tissue. “In designing panels for IMC, we apply the same biological considerations that one considers in suspension-based mass cytometry. Due to the large heterogeneity in cell types in imaging, this can make panel design simpler than in suspension,” says Quong.

Like suspension-based mass cytometry, IMC has a transcriptomic analog: spatial transcriptomics. “The primary advantage of IMC over spatial transcriptomics is that IMC is a true imaging technology that has sufficient resolution to determine the specific locations of single cells,” adds Quong.

Indeed, the resolution of spatial transcriptomics pales in comparison to that of IMC. 10X Genomic’s Visium Arrays, for example, have a worse resolution than the naked eye (55–100 vs. 40 μm).^2,3 Only very recently did spatial transcriptomics catch up to IMC in terms of resolution with the advent of Seq-Scope.³ But this technology is not a true imaging technology; rather, it’s a variation on in situ sequencing that uses Illumina sequencing-by-synthesis to read spatial information from barcoded probes. As such, the method is only readily accessible to a select few who possess the requisite expertise and capabilities.

Its ability to interrogate highly dimensional cell populations with single-cell resolution makes IMC a good fit for many applications, even beyond the confines of the brain. “This idea of being able to look at the spatial relationships of 30 or so markers and the cells they’re expressed on can help us build a better understanding of everything from cellular development to cancer biology,” says Bushnell.

References

1. Baskar R, Kimmey SC, Bendall SC. Revealing new biology from multiplexed, metal-isotope-tagged, single-cell readouts [published online ahead of print, 2022 Feb 16]. Trends Cell Biol. 2022;S0962-8924(22)00027-7. 

2. Bergenstråhle J, Larsson L, Lundeberg J. Seamless integration of image and molecular analysis for spatial transcriptomics workflows. BMC Genomics. 2020;21(1):482. Published 2020 Jul 14. 

3. Cho CS, Xi J, Si Y, et al. Microscopic examination of spatial transcriptome using Seq-Scope. Cell. 2021;184(13):3559-3572.e22.