Mass cytometry, also known as cytometry by time-of-flight (CyTOF), fuses the experimental platforms of flow cytometry and elemental mass spectrometry. Mass cytometers require antibodies conjugated to rare earth metal isotopes of defined atomic mass instead of fluorochromes. Although throughput and sensitivity is less than conventional flow cytometry (10x less), the unique mass signatures of these stable metal isotopes by time-of-flight mass spectrometry (TOFMS) improves specificity by accurately discriminating metal isotopes of different atomic masses without the need for isotype controls. This unique signature of metals provides simultaneous identification of over 40 targets per cell, providing greater capabilities of phenotyping cells than flow cytometry (with just over 20 parameters using multi-color panels).
Mass Cytometry Basics
To quantify the expression level of proteins on individual cells, metal isotopes can be conjugated to primary antibodies for mass cytometry analysis. Fixed, stained cells are passed in a single-cell suspension into a nebulizer as droplets into the mass cytometer. Cells are then injected into an argon flow chamber and exposed to a plasma torch, vaporizing cells into an ionized cloud. The ion clouds are passed through a quadrupole to enrich the heavy-metal reporter ion while removing impurities. Ions are separated by their mass-to-charge ratio in a time-of-flight mass spectrometer. Cell particles come out according to the conjugated atomic mass, where heavier mass-to-charge metal tags have a longer time-of-flight. Every column represents a distinct isotope measured and each row represents a single mass scan of the detector.
Antibodies conjugated to metal isotopes
Single-cell suspension carried into nebulizer of the mass cytometer
Cells injected into an argon flow chamber are exposed to a plasma torch
Metal tagged vaporized cells form ion clouds sent through Quadrupole for purification and concentration.
Times of flight (TOFs) of the ions from the plasma are measured by a mass spectrometer detector.
Individualized cells are profiled by atomic mass (i.e. lighter metals arrive first)
Advantages Over Fluorescence Flow Cytometry
Reduce Spectral Overlap: TOFMS accurately discriminates metal isotopes of different atomic masses without channel overlap.
Minimal compensation: No need for fluorescence compensation and minimal background observed from the instrument if cleaned properly.
Eliminate Autofluorescence Background: Absence of interference from cellular autofluorescence as cells do not contain metals intrinsically.
Panel design: 40+ channels available in CyTOF, increasing flexibility and ease in panel design.
Improve Molecular Profiling: Simultaneous detection and resolution of more targets than is feasible with conventional flow cytometry, making TOFMS ideal for testing rare or precious samples.
Bio-Techne CyTOF Antibodies
R&D Systems and Novus Biologicals have a wide range of antibodies that are bottled BSA and azide-free in a CyTOF-ready formulation for metal conjugation. Additional benefits for established conjugation methods include:
Liquid formulation or lyophilized for reconstitution at high concentration and improved conjugation efficiency.
No need for a buffer exchange step prior to conjugation.
Over 2800 CyTOF-ready antibodies available.
Also available conjugated to fluorochromes for fluorescence flow cytometry.
Antibody conjugation: Metal conjugation antibody kits can be laborious and require meticulous attention to detail.
Consumables cost: The use of rare earth metals in metal labeling antibody kits and required use of inert gases for all experiments make up a significant consumables cost.
Slower acquisition: Compared to fluorescence flow cytometry, CyTOF experiments run at a slower rate (1,000 cells/sec vs 10,000 cells/sec). This leads to increased costs for instrument use and therefore can become cost prohibitive. However, the costs are offset by the capability of CyTOF to do extensive molecular profiling, which produces more data from each cell.
No downstream analysis: Due to the nature of the application, cells are vaporized into an ionized cloud, losing the capabilities to sort cells for further analysis or downstream applications.
Imaging Mass Cytometry
Further advanced technology known as imaging mass cytometry fuses immunohistochemistry and immunocytochemistry imaging techniques with mass cytometry. In imaging mass cytometry, metal conjugated antibodies are used on paraffin-embedded tissue sections. Laser ablation separates cells from the fixed tissue slide (fresh or frozen) and the volatized cells are carried by inert gas into the mass cytometer for analysis described above.
Mass Cytometry Webinar
In the Bio-Techne webinar, “Single-Cell Profiling with Mass Cytometry: An Overview of Technology and Current Research Applications On-Demand”, learn more about CyTOF from a leading expert.
Regulation of adaptive NK cells and CD8 T cells by HLA-C correlates with allogeneic hematopoietic cell transplantation and with cytomegalovirus reactivation. Horowitz, A. et al. J. Immunol. (2015) 195:4524. (Catalog # MAB138, MAB1844, MAB2014)