Abbreviations:CyTOF (mass cytometry by time-of-flight), MCA (metal-conjugated antibody), MCB (mass-tag cellular barcoding)
- •Recognize the newest techniques in biomedical research.
- •Describe how these techniques can be utilized and their limitations.
- •Describe the potential impact of these techniques.
- •The success of mass cytometry experiments is dependent on well–thought-out goals, a detailed experimental design, and practice with CyTOF technology and protocols.
- •When designing custom metal-conjugated antibody (MCA) panels, account for target antigen abundance and signal crosstalk.
- •Custom MCAs need to be thoroughly validated and titrated.
- •Staining protocols may need to be tested to optimize marker signal detection. Metal nucleic acid intercalators should be included in the experiment for accurate single-cell identification and live:dead discrimination.
- •To minimize sample-to-sample variation, it is important to normalize samples based on bead standards and/or a sample barcoding strategy.
Metal-Conjugated Antibody Panel Design
|Factors to account for signal detection with minimum background|
System sensitivity to metal isotope
Background signal (environment)
Crosstalk (abundance sensitivity, metal isotope purity, metal ion oxidation)
Mass-Tag Cellular Barcoding
Conflict of Interest
Multiple Choice Questions
- 1.Which of the following are two main considerations when designing a CyTOF panel?
- A.Antigen abundance and crosstalk
- B.Use only ready-to-use panel kits and commercially available MCAs
- C.Low number of available probes and sample cell type
- D.Vast variability of signal detection across channels and low isotopic purity
- 2.What are the principal sources of signal background in CyTOF?
- A.Concentration of metal-conjugated antibodies and reagents during staining
- B.Environmental contamination and crosstalk
- C.Plasma temperature and instrument state of maintenance
- D.Endogenous cell signal and spectral overlap
- 3.Which cell-staining protocol should be used for CyTOF experiments?
- A.Standard CyTOF protocol
- B.Same protocol as for flow cytometry
- C.CyTOF staining protocol tested for specific experiment panel
- D.Any CyTOF-validated protocol
- 4.What can be done to ensure an accurate analysis of live single cells?
- A.Use only fresh and filtered cell samples.
- B.Use nucleic acid intercalators.
- C.Acquire data on the same day as performing the staining protocol.
- D.Use calibration beads.
- 5.Which CyTOF-specific strategies should be used to control for sample-to-sample variation?
- A.Use a commercial, ready-to-use panel kit and high-quality reagents.
- B.Incorporate nucleic acid intercalators and use the same cell concentration in all samples.
- C.Make sure to use appropriate statistical tests and the same analysis software for all samples.
- D.Normalize samples based on bead standards and barcode samples.
- Quiz and brief explanation of correct answers
- Teaching Slides
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- Single cell mass cytometry of differential immune and drug responses across the human hematopoietic continuum.Science. 2011; 332: 687-696
- Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.Cytometry. 2016; 89: 491-497
- Flow cytometry II: mass and imaging cytometry.J Invest Dermatol. 2015; 135: e36
- Normalization of mass cytometry data with bead standards.Cytometry A. 2013; 83: 483-494
- Two functional subsets of FOXP3+ regulatory T cells in human thymus and periphery.Immunity. 2008; 28: 870-880
- A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).Cytometry A. 2015; 87: 369
- Polymer-based elemental tags for sensitive bioassays.Angew Chem Int Ed. 2007; 46: 6111-6114
- Mass cytometry: single cells, many features.Cell. 2016; 165: 780-791
- Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.Cytometry A. 2015; 87: 357-368
- Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.Sci Transl Med. 2015; 7: 279ra39
- Mapping the diversity of follicular helper T cells in human blood and tonsils using high dimensional mass cytometry analysis.Cell Reports. 2015; 11: 1822
- Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.Nat Protocols. 2015; 10: 316-333
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