Research Techniques Made Simple: Experimental Methodology for Single-Cell Mass Cytometry

  • Tiago R. Matos
    Correspondence
    Correspondence: Tiago R. Matos, Department of Dermatology, Room L3-119, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
    Affiliations
    Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Harvard Medical School, Boston, Massachusetts, USA

    Academic Medical Center, Department of Dermatology, University of Amsterdam, Amsterdam, The Netherlands
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  • Hongye Liu
    Affiliations
    Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Harvard Medical School, Boston, Massachusetts, USA
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  • Jerome Ritz
    Affiliations
    Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Harvard Medical School, Boston, Massachusetts, USA
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  • Author Footnotes
    a The abbreviation “CyTOF”, in addition to being the name of this technique, is also the name of a commercial product that enables researchers to use the method. The authors are in no way endorsing any specific commercial products
      Growing recognition of the complexity of interactions within cellular systems has fueled the development of mass cytometry. The precision of time-of-flight mass spectrometry combined with the labeling of specific ligands with mass tags enables detection and quantification of more than 40 markers at a single-cell resolution. The 135 available detection channels allow simultaneous study of additional characteristics of complex biological systems across millions of cells. Cutting-edge mass cytometry by time-of-flight (CyTOF) can profoundly affect our knowledge of cell population heterogeneity and hierarchy, cellular state, multiplexed signaling pathways, proteolysis products, and mRNA transcripts. Although CyTOF is currently scarcely used within the field of investigative dermatology, we aim to highlight CyTOF’s utility and demystify the technique. CyTOF may, for example, uncover the immunological heterogeneity and differentiation of Langerhans cells, delineate the signaling pathways responsible for each phase of the hair cycle, or elucidate which proteolysis products from keratinocytes promote skin inflammation. However, the success of mass cytometry experiments depends on fully understanding the methods and how to control for variations when making comparisons between samples. Here, we review key experimental methods for CyTOF that enable accurate data acquisition by optimizing signal detection and minimizing background noise and sample-to-sample variation.

      Abbreviations:

      CyTOF (mass cytometry by time-of-flight), MCA (metal-conjugated antibody), MCB (mass-tag cellular barcoding)
      CME Activity Dates: 21 March 2017
      Expiration Date: 20 March 2018
      Estimated Time to Complete: 1 hour
      Planning Committee/Speaker Disclosure: All authors, planning committee members, CME committee members and staff involved with this activity as content validation reviewers have no financial relationship(s) with commercial interests to disclose relative to the content of this CME activity.
      Commercial Support Acknowledgment: This CME activity is supported by an educational grant from Lilly USA, LLC.
      Description: This article, designed for dermatologists, residents, fellows, and related healthcare providers, seeks to reduce the growing divide between dermatology clinical practice and the basic science/current research methodologies on which many diagnostic and therapeutic advances are built.
      Objectives: At the conclusion of this activity, learners should be better able to:
      • Recognize the newest techniques in biomedical research.
      • Describe how these techniques can be utilized and their limitations.
      • Describe the potential impact of these techniques.
      CME Accreditation and Credit Designation: This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint providership of William Beaumont Hospital and the Society for Investigative Dermatology. William Beaumont Hospital is accredited by the ACCME to provide continuing medical education for physicians.
      William Beaumont Hospital designates this enduring material for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      Method of Physician Participation in Learning Process: The content can be read from the Journal of Investigative Dermatology website: http://www.jidonline.org/current. Tests for CME credits may only be submitted online at https://beaumont.cloud-cme.com/RTMS-Apr17 – click ‘CME on Demand’ and locate the article to complete the test. Fax or other copies will not be accepted. To receive credits, learners must review the CME accreditation information; view the entire article, complete the post-test with a minimum performance level of 60%; and complete the online evaluation form in order to claim CME credit. The CME credit code for this activity is: 21310. For questions about CME credit email [email protected] .

      Introduction

      Growing recognition of the complexity of interactions within cellular systems has fueled development of new technologies capable of a broad, holistic scope of analysis. In mass cytometry by time-of-flight (CyTOF)
      The abbreviation “CyTOF”, in addition to being the name of this technique, is also the name of a commercial product that enables researchers to use the method. The authors are in no way endorsing any specific commercial products
      , cells are probed with metal-conjugated antibodies (MCAs). Tagged cell suspensions are then passed through a droplet nebulizer to enter into argon plasma, where individual cells are atomized and ionized, and abundant common ions are removed. Then, time-of-flight mass spectrometry detects the ionized metal tags through 135 detection channels and can measure over 45 parameters in each cell (
      • Bandura D.R.
      • Baranov V.I.
      • Ornatsky O.I.
      • Antonov A.
      • Kinach R.
      • Lou X.
      • et al.
      Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.
      ) (Table 1). Mass cytometry has been described in detail in previous reviews (
      • Bendall S.C.
      • Nolan G.P.
      • Roederer M.
      • Chattopadhyay P.K.
      A deep profiler’s guide to cytometry.
      ,
      • Doan H.
      • Chinn G.M.
      • Jahan-Tigh R.R.
      Flow cytometry II: mass and imaging cytometry.
      ). Despite the innovative applications of this technique, it is currently scarcely used within the field of investigative dermatology. CyTOF technology may lead to a greater comprehension of cutaneous cellular phenotype heterogeneity, development, hierarchy, and relationship to other tissues. CyTOF may allow simultaneous study of cell state (such as proliferation, hypoxia, or enzymatic activity) and deeper understanding of expression of mRNA transcripts, cytokines, growth factors, or transcription factors within cell subsets. Examples of questions that may be addressed using this technique are abundant. Which signaling pathways of innate lymphoid cells effectively regulate immune homeostasis or contribute to autoimmunity? Which proteolysis products of keratinocytes promote skin inflammation? Which cancer cells have predictive value for early diagnosis, prognosis, development of drug resistance, or relapse?
      Table 1Summary description of mass cytometry technology, advantages and limitations
      AdvantagesLimitations
      • Possible to simultaneously analyze over 45 parameters (e.g., 40 antibody-tagged markers, cell viability, and DNA content)
      • Possible to study cell death, cytokine production, and cell signaling simultaneously
      • Minimal background noise from signal overlap or endogenous cellular components
      • Cost per probe per test ≈ $1.50–$3.00
        Estimated based on the price of commercially conjugated reagents or unconjugated antibodies and commercial conjugation kits, in contrast to $2.00–$8.00 for fluorescent flow cytometry (Bendall et al., 2012).
      • Cost per analyzed cell ≈ 0.005 cents
        The cost of reagents, disposables, and data acquisition, in contrast to ∼$22 to measure cells by single-cell RNA sequencing using the Fluidigm C1 system (Fluidigm, San Francisco, CA) and molecular identifiers (Spitzer et al., 2016).
      • A single dataset can be analyzed simultaneously by various analysis methods to test multiparameter hypotheses
      • Cells are destroyed through the CyTOF process; thus, it is not feasible to further culture or analyze cells after data acquisition
        Slow sample throughput (maximum of 2,000 events/second), whereas flow cytometry can operate 25–50 times faster
      • Some cellular properties cannot be measured (e.g., pH or ion concentration)
      • Low efficiency (only 30–60% of cells of a sample are measured)
      • CyTOF’s multiplexed, high-dimension data requires new analysis tools
      Abbreviation: CyTOF, mass cytometry by time-of-flight.
      CyTOF allows the characterization and quantification of over 40 markers simultaneously on millions of individual cells. Metal-tagged antibodies are used to label multiple internal and external cellular markers of interest, which can be quantified by time-of-flight mass spectrometry at a single-cell resolution. It can lead to unprecedented breakthroughs of understanding the complex differentiation process and interaction between cell subpopulations, new cell types, functional profiles, and biomarkers.
      1 Estimated based on the price of commercially conjugated reagents or unconjugated antibodies and commercial conjugation kits, in contrast to $2.00–$8.00 for fluorescent flow cytometry (
      • Bendall S.C.
      • Nolan G.P.
      • Roederer M.
      • Chattopadhyay P.K.
      A deep profiler’s guide to cytometry.
      ).
      2 The cost of reagents, disposables, and data acquisition, in contrast to ∼$22 to measure cells by single-cell RNA sequencing using the Fluidigm C1 system (Fluidigm, San Francisco, CA) and molecular identifiers (
      • Spitzer M.H.
      • Nolan G.P.
      Mass cytometry: single cells, many features.
      ).
      This review aims to highlight CyTOF’s novelty and utility, demystify the technique, and provide guidance on design of the multistep experimental methodology that requires detailed understanding and planning to ensure accurate and consistent results. We will focus on specific considerations needed when designing a panel of desired markers, optimizing the staining protocol, performing metal conjugation of antibodies, and barcoding multiple samples.

      Summary Points

      • 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.

      Experimental Design

      Mass cytometry experiments include precise and lengthy multistep protocols that generate immense amounts of data at single-cell resolution (Figure 1). It is therefore imperative to establish a meticulous experimental strategy and to have clear objectives at the onset of each experiment. After defining specific experimental aims, it is important to define the types of cells that will be studied, experimental conditions, comparative groups, and controls. In some cases, FACS or magnetic-activated cell sorting is needed to enrich for rare subsets of cells to avoid long CyTOF acquisition times (sample throughput: flow cytometry = 25,000 vs. CyTOF = 500–2,000 cells/second). At least 300 events of the rarest population should be acquired for analysis.
      Figure 1
      Figure 1Schematic representation of mass cytometry experiment design. See text for details.
      • Watanabe R.
      • Gehad A.
      • Yang C.
      • Scott L.L.
      • Teague J.E.
      • Schlapbach C.
      • et al.
      Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.
      recently used mass cytometry to compare the relative functional capacities (including TNF-α, IL-2, IL-4, IL-13, IFN-γ, IL-17, IL-22, and IL-10) of skin-tropic (CLA+) central memory, migratory memory, and effector memory T cells from human blood. T cells were isolated from peripheral blood of healthy individuals and stimulated with phorbol 12-myristate 13-acetate and ionomycin. The opportunity to simultaneously study those many markers from a single sample enabled the authors to conclude that effector memory T cells secrete more T helper type 1, T helper type 17, and T helper type 22 proinflammatory effector cytokines, whereas central memory T cells have higher T helper type 2 cytokine production.
      It is important to start an experimental design by listing the markers necessary to define the cell populations that will be studied. Further markers can then be added to the panel for specific experimental measurements. Complementary staining panels can even be combined to study more than 40 markers from the same sample. For example,
      • Bendall S.C.
      • Simonds E.F.
      • Qiu P.
      • Amir E.D.
      • Krutzik P.O.
      • Finck R.
      • et al.
      Single cell mass cytometry of differential immune and drug responses across the human hematopoietic continuum.
      aimed to develop an immune system map of healthy human bone marrow cells, allowing comparisons with bone marrow cells after drug treatment or in cases of disease. This could allow mechanistic studies and pharmacologic intervention. The authors wanted to simultaneously analyze 52 different single-cell parameters, which are more markers than either CyTOF (∼40) or flow cytometry (∼15–20) can detect at a single time. Two complementary staining panels were used for the same samples. (Each panel had the same 16 core markers and an additional 18 panel-specific markers). Merging the data allowed analysis of the biochemical intracellular signaling in rare and diverse subsets of human bone marrow cells.
      A recent study showed that each CyTOF instrument has its own signal sensitivity profile for metal isotopes, suggesting that it may be difficult to accurately compare data acquired by different instruments. This should be taken into account in experimental design, especially for multicenter studies using different CyTOF instruments (
      • Tricot S.
      • Meyrand M.
      • Sammicheli C.
      • Elhmouzi-Younes J.
      • Corneau A.
      • Berholet S.
      • et al.
      Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.
      ). Even though it is currently possible to normalize variation over time relative to a single CyTOF instrument by including calibration beads with each run, it is not yet possible to normalize data acquired by different instruments. An alternative is to include a standard/control sample (having several vials of the same sample frozen) together with each run of the samples of interest. Each sample’s data can later be normalized according to the control sample that was analyzed within that same run. It has also been suggested that beads should be embedded with elements representing the complete detectable mass range to allow an algorithm to be developed to normalize output for each channel and instrument (
      • Tricot S.
      • Meyrand M.
      • Sammicheli C.
      • Elhmouzi-Younes J.
      • Corneau A.
      • Berholet S.
      • et al.
      Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.
      ).

      Metal-Conjugated Antibody Panel Design

      Design of mass cytometry panels requires the assignment of a distinct metal isotope to each of the antibodies from the list of desired markers. There are several commercially available, ready-to-use panels that have been designed to study distinct cell types and functional systems. However, when customizing panels by adding new markers or designing a new panel, careful planning and awareness of multiple factors that can influence signal acquisition are needed to optimize signal detection and minimize background noise (Table 2). The nature of the current CyTOF mass window results in an optimal detection of metals in the range of 153–176 Da. Therefore, low-abundance targets should be tagged with metals within this mass range (Figure 2). In CyTOF, the main sources of background noise come from environmental contamination with untagged metals or the measurement of the isotope signal in undesired channels, referred to as crosstalk. Environmental contamination of samples can drastically compromise data acquisition. This is reduced by the use of water purified by reverse osmosis (e.g., Milli Q water; Merck Millipore, Darmstadt, Germany) and certified metal-free buffers and containers. Sources of crosstalk include oxidation, isotopic purity, and signal abundance sensitivity. Several ions oxidize at low frequencies (≤0.1–3.0%), resulting in a mass gain that is then read by the corresponding channel. Oxidation can be minimized by optimizing the plasma temperature, which is part of the daily CyTOF tuning procedure. Isotopic purity is generally high (95–99%), but nevertheless, the signal from residual metal isotopes can bleed and be read by corresponding channels. Online tools are available to help researchers minimize crosstalk and optimize study-specific MCAs.
      Table 2Factors that can influence signal detection
      Factors to account for signal detection with minimum background
      Antigen abundance

      System sensitivity to metal isotope

      Background signal (environment)

      Crosstalk (abundance sensitivity, metal isotope purity, metal ion oxidation)
      Figure 2
      Figure 2Metal-conjugated antibody panel design. Example of mass response curve with optimal detection of metals in the range of 153–176 Da (pink bar).

      Metal-Antibody Conjugation

      Over 500 metal conjugated antibodies are commercially available to study human and mouse cells. It is also possible to conjugate in-house additional IgG antibodies to one of the existing 36 metal isotopes or request a custom conjugation service, available through commercial vendors or institutional core facilities (
      • Lou X.
      • Zhang G.
      • Herrera I.
      • Kinach R.
      • Ornatsky O.
      • Baranov V.
      • et al.
      Polymer-based elemental tags for sensitive bioassays.
      ). Each new custom MCA should be validated with relevant positive and negative control cell lines with known expression of the marker being tested. It is also often necessary to compare the results acquired by CyTOF with conventional flow cytometry, using a fluorochrome-tagged antibody identical to the purified clone used for the metal-antibody conjugation (Figure 3). To avoid ion detector saturation, new MCA and commercial ready-to-use MCA concentrations must be titrated. To facilitate these comparisons it is useful to store several vials of the same target cell population that can be thawed and used as a control, because then the expected expression of a specific marker would already be known from previous tests. When purchasing custom MCAs it is important to know what validations and titrations were conducted, to determine whether these are relevant to the specific research project at hand.
      Figure 3
      Figure 3Example of validation of custom metal antibody conjugation (ICOS-154Sm). (a) An initial validation of custom metal-conjugated antibodies can be carried out by assessing the expression of the marker in previously known populations (e.g., by searching for information published in the literature;
      • Ito T.
      • Hanabuchi S.
      • Wang Y.H.
      • Park W.R.
      • Arima K.
      • Bover L.
      • et al.
      Two functional subsets of FOXP3+ regulatory T cells in human thymus and periphery.
      ). (b) Results from identical cell samples analyzed by CyTOF and flow cytometry can be compared for a limited number of markers. Here, a fluorochrome-tagged antibody identical to the purified clone used for the metal-antibody conjugation was used for FACS. Alternatively, positive and negative control cell lines should be used. DNA2 stands for iridium-193. CyTOF, mass cytometry by time-of-flight; SSC, side scatter.

      Cell-Staining Protocol

      The cell-staining protocol can also depend on the experimental goals; for example, consideration should be given to whether cells need to be stimulated or whether the study involves cells surface markers, intercellular markers (cytokines and chemokines), intranuclear markers (transcription factors), or tetrameters (T-cell specificity). Online resources, for example at http://web.stanford.edu/group/nolan, offer CyTOF-validated protocols for cell staining. Protocols must occasionally be slightly adjusted to optimize signal sensitivity. For instance, a possible adjustment may be due to the down-regulation of some surface markers (e.g., chemokine receptors) when cells are stimulated.
      • Wong M.T.
      • Chen J.
      • Narayanan S.
      • Lin W.
      • Anicete R.
      • Kiaang H.T.
      • et al.
      Mapping the diversity of follicular helper T cells in human blood and tonsils using high dimensional mass cytometry analysis.
      noticed a 10% difference in frequency of CXCR5 expression on tonsillar and peripheral CD4+ T cells upon phorbol 12-myristate 13-acetate/ionomycin stimulation, which was prevented by including metal-tag antibodies against the trafficking receptors (i.e., CXCR5), together with the stimulation medium. This is likely because metal-tag antibodies have a higher antibody conjugate stability compared with fluorophore-tag antibodies during cell incubation (
      • Wong M.T.
      • Chen J.
      • Narayanan S.
      • Lin W.
      • Anicete R.
      • Kiaang H.T.
      • et al.
      Mapping the diversity of follicular helper T cells in human blood and tonsils using high dimensional mass cytometry analysis.
      ).
      If necessary to achieve a better separation between cell subsets expressing or not expressing a marker, a secondary antibody can be used (e.g., anti–Ki67-PE followed by anti–PE-metal) to amplify the signal. Some antigens can be tagged using either surface or intracellular staining protocols (e.g., CTLA-4), resulting in different signal intensities. In our experience, protocol adjustments are based on studying the same markers by traditional flow cytometry or by testing distinct staining protocols side by side. Because protocol optimization may take several rounds of trial and error, we use control samples (e.g., cell lines or healthy control samples) before analyzing precious experimental samples.
      For discrimination between live and dead cells, rhodium 103 nucleic acid intercalators or cisplatin (platinum-195, -194, or -198) should be included in the experimental protocol. Rhodium and cisplatin label only dead cells, because they have compromised cell membranes. Resulting data can then be gated on rhodium-negative or cisplatin-negative cells to ensure analysis of live cells only.
      For accurate single-cell identification, cells can be stained with two distinct iridium isotopes (iridium-191 and iridium-193) after fixation and permeabilization. Iridium intercalates DNA with high affinity, allowing detection of all DNA-containing cells and aiding the identification of single-cell events (excludes doublets).

      Mass-Tag Cellular Barcoding

      Using a newly developed mass-tag cellular barcoding (MCB) strategy, it is possible to individually barcode up to 20 samples that can then be processed together in a single tube. After data acquisition, software deconvolutes samples based on the barcode present in each cell, allowing the analysis and comparison of each original sample separately. It guarantees consistent quality of signal detection and better efficiency of data acquisition by reducing the time needed to wash the instrument between samples. When the MCB is done before cell staining, it also reduces antibody consumption and staining protocol workflow time and adds consistency throughout sample processing and staining that is crucial to providing accurate comparisons among samples.
      Each sample can be barcoded with a unique combination of two or three of six available palladium isotopes (Figure 4). Alternatively, osmium and ruthenium tetroxides (OsO4 and RuO4), which bind covalently with fatty acids in cellular membranes and aromatic amino acids in proteins, can be used independently or combined with palladium isotopes for MCB of cells (
      • Catena R.
      • Özcan A.
      • Zivanovic N.
      • Bodenmiller B.
      Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.
      ). When studying peripheral blood mononuclear cells, it is also possible to use six distinct anti-CD45 antibodies conjugated with different metal isotopes (
      • Lai L.
      • Ong R.
      • Li J.
      • Albani S.
      A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).
      ).
      Figure 4
      Figure 4Single-cell barcode and deconvolution. (a) Each sample is barcoded with a unique combination of three of six available palladium (Pd) isotopes. (b) Five events from a 6-choose-3 mass-tag cellular barcoding (MCB)-multiplexed fluorescent cell standard (FCS) file are shown in single-cell format displayed on a vertical dashed line. Events 1, 2, and 3 correspond to barcode single cells as indicated by the isotope symbols, Event 4 is a barcode doublet, and Event 5 is classified as debris. The red line segments indicate barcode separation, assuming the 6-choose-3 scheme, which is always set as the distance between the third and fourth highest barcode intensities. Without this assumption, the last two events would have larger barcode separations but would still be discarded because their barcodes would not match any in the 20-sample scheme. (c) Examples of a barcode singlet (three positive barcode channels) and a barcode doublet (more than three positive barcode channels) as seen in the time-of-flight spectra used to visualize cells while acquiring data at the instrument. ms, mass spectra.
      Adapted with permission from Macmillan Publishers Ltd:
      • Zunder E.R.
      • Finck R.
      • Behbehani G.K.
      • Amir el-AD.
      • Krishnaswamy S.
      • Gonzalez V.D.
      • et al.
      Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.
      .
      Some drawbacks of MCB are that cell yields can be reduced by up to 50% after barcoding. There are several factors that can influence cell loss, such as low initial cell number, cell type, container type, procedures for liquid transfer, and mixing. One MCB protocol put forth by
      • Zunder E.R.
      • Finck R.
      • Behbehani G.K.
      • Amir el-AD.
      • Krishnaswamy S.
      • Gonzalez V.D.
      • et al.
      Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.
      includes an early sample pooling step, which creates a single high-abundance sample rather than multiple low-abundance samples, improving the cell yield. Additionally, MCB requires fixation and permeabilization of cells that, when done before antibody staining, may affect the ability to detect some epitopes. Epitopes that are sensitive to the paraformaldehyde fixation step should be stained with antibodies before barcoding. Alternatively, it is necessary to identify a paraformaldehyde-compatible epitope for the same marker of interest (
      • Behbehani G.K.
      • Thom C.
      • Zunder E.R.
      • Finck R.
      • Gaudilliere B.
      • Fragiadakis G.K.
      • et al.
      Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.
      ,
      • Zunder E.R.
      • Finck R.
      • Behbehani G.K.
      • Amir el-AD.
      • Krishnaswamy S.
      • Gonzalez V.D.
      • et al.
      Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.
      ) (Table 3).
      Table 3Advantages and limitations of mass-tag barcoding
      AdvantagesLimitations
      • Reduces technical variability (cell-to-antibody ratio-dependent effects on staining, pipetting errors, sample acquisition, etc.)
      • Reduces antibody consumption, staining protocol workflow time, and data acquisition time
      • Allows staining of valuable samples with low cell numbers by combining numerous samples together
      • Excludes debris and cross-sample doublets (e.g., cell events with more or less than three palladium isotopes)
      • Barcoding reagents can be costly
      • Low cell yield (∼50%)
      • Barcoding method can affect quality of antibody staining for some epitopes

      Calibration Beads

      To minimize the variance in mass cytometry instrument performance over time that may produce sample-to-sample signal variation, calibration beads should be added to the sample immediately before the sample is injected into the instrument for CyTOF acquisition. After acquisition, data normalization can be carried out using an algorithm to analyze the calibration bead signal variation that was captured simultaneously with the cell samples, enabling correction of both short- and long-term signal fluctuations (
      • Finck R.
      • Simonds E.F.
      • Jager A.
      • Krishnaswamy S.
      • Sachs K.
      • Fantl W.
      • et al.
      Normalization of mass cytometry data with bead standards.
      ). This normalization algorithm is currently part of the general CyTOF software package.

      Conclusion

      The success of mass cytometry-based experiments is dependent on well–thought-out goals, detailed experimental design, and knowledge of potential technical pitfalls and limitations. A methodical approach is essential to control for experimental noise when conducting precise comparisons between samples. This approach facilitates the ability to harness the full potential of mass cytometry to characterize complex biological systems at single-cell resolution. CyTOF may lead to key discoveries in investigative dermatology, including identification of signaling phenotypes with predictive value for early diagnosis, prognosis, or relapse, and a thorough characterization of intratumor heterogeneity and disease-resistant cell populations that may ultimately unveil novel therapeutic approaches.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We would like to thank Jodi L. Johnson for helpful comments, critical reading of the manuscript, and editorial assistance. Funded by the Harvard Medical School–Portugal Program in Translational Research HMSP-ICT/0001/201.

      CME Accreditation

      This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint providership of William Beaumont Hospital and the Society for Investigative Dermatology. William Beaumont Hospital is accredited by the ACCME to provide continuing medical education for physicians. William Beaumont Hospital designates this enduring material for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      To participate in the CE activity, follow the link provided.
      https://beaumont.cloud-cme.com/RTMS-Apr17

      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.

      Supplementary Material

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