Development of a Click-Chemistry Reagent Compatible with Mass Cytometry

Shaklee, Jessica; Srivastava, Kriti; Brown, Heather M.; Arriaga, Edgar A.; Pierre, Valérie C.; Berlo, Jop H. van

doi:10.1038/s41598-018-25000-y

Cited by 4 publications

(10 citation statements)

References 48 publications

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“…32,33 Previously, Ho-DOTA was used to demonstrate that a nonspecific background was low in the cell populations that were monitored for DNA replication with a "clickable" chemistry probe. 19 Past works revealed population level trends and did not explore the significance of non-specific retention correction in single cells. Inspired by our previous work using antibody isotypes to make corrections for non-specific binding in each cell; 34 here, we simultaneously treated cells with Tb-DOTAazide and Ho-DOTA (Figure 1C) to develop a novel method for correction for non-specific signals in each single cell.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The mass cytometry field has seen an increase in alternative reporters to antibodies including those for RNA, 17 cell cycle analysis, 18 DNA synthesis, 19 aptamers, 20 and enzymatic activity. 21 In addition to the expansion of reporters, alternative chemistries have been applied such as click chemistry and the use of nanoparticles to expand the capabilities of mass cytometry.…”

mentioning

confidence: 99%

“…21 In addition to the expansion of reporters, alternative chemistries have been applied such as click chemistry and the use of nanoparticles to expand the capabilities of mass cytometry. 19,22,23 Click chemistry has been applied as an alternative linker chemistry in metal tagging antibodies, peptides, and lectins and as a cell treatment for tracking DNA synthesis using an alkyne containing EdU and a terbiumchelated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-azide reporter. 24 Here, we report a strategy to track prenylation along with molecular markers detected with antibody reporters in mass cytometric analysis of single cells.…”

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confidence: 99%

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Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells

et al. 2022

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Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer’s disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide–alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.

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Section: ■ Results and Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells

et al. 2022

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“…In a separate experiment, we administered the thymidine analog 5-Ethynyl-2'-deoxyuridine (EdU) to mice before isolating fixed cardiomyocytes. After cardiomyocyte isolation, we stained for incorporated EdU using standard protocols 13 , and were able to detect cardiomyocytes that had undergone S phase in either mononucleated, binucleated and trinucleated cardiomyocytes ( Figure 2B).…”

Section: Representative Resultsmentioning

confidence: 99%

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

Yücel

Solinsky

Berlo

2020

JoVE

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The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and α-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

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“…Marker screen kits are available ( 68 , 69 ). Beyond isotope-conjugated antibodies, other probes which can be included in mass cytometry panels include tetramers ( 70 ), carbohydrate-binding molecules ( 71 ), tellurium-based oxygen sensors ( 72 ), inorganic nanoparticles ( 73 ), RNA probes ( 74 , 75 ), and modified nucleotides ( 75 , 76 ) ( Figure 2A ). With the exception of oxygen ( 72 ), small molecules or proteins were not detectable by mass cytometry until the Nitz group developed a Tellurium-containing analog of phenylalanine, making it possible to monitor protein synthesis ( 77 ).…”

Section: Panel Design and Antibody Conjugationmentioning

confidence: 99%

CyTOF® for the Masses

2022

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Mass cytometry has revolutionized immunophenotyping, particularly in exploratory settings where simultaneous breadth and depth of characterization of immune populations is needed with limited samples such as in preclinical and clinical tumor immunotherapy. Mass cytometry is also a powerful tool for single-cell immunological assays, especially for complex and simultaneous characterization of diverse intratumoral immune subsets or immunotherapeutic cell populations. Through the elimination of spectral overlap seen in optical flow cytometry by replacement of fluorescent labels with metal isotopes, mass cytometry allows, on average, robust analysis of 60 individual parameters simultaneously. This is, however, associated with significantly increased complexity in the design, execution, and interpretation of mass cytometry experiments. To address the key pitfalls associated with the fragmentation, complexity, and analysis of data in mass cytometry for immunologists who are novices to these techniques, we have developed a comprehensive resource guide. Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and streamlined project workflow. It is our hope that this manuscript will prove a useful resource for both beginning and advanced users of mass cytometry.

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Development of a Click-Chemistry Reagent Compatible with Mass Cytometry

Cited by 4 publications

References 48 publications

Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells

Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

CyTOF® for the Masses

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