Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry

Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Antonov, A.; Kinach, Robert; Lou, Xudong; Pavlov, Serguei; Vorobiev, S.A.; Dick, John E.; Tanner, Scott D.

doi:10.1021/ac901049w

Cited by 1,130 publications

(941 citation statements)

References 29 publications

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“…To test this hypothesis we investigated six intracellular cytokines on MAIT cells ex vivo in multiparametric analysis using mass cytometry 33,34 MIP-1b, IFN-g, TNF-a and IL-2 (Fig. 4c,d), whereas IL-4 þ and IL-10 þ cells were less frequent.…”

Section: Cdr3b Length Distribution Differs In Mait and Non-mait Cellsmentioning

confidence: 99%

Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire

et al. 2014

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Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize conserved bacterial antigens derived from riboflavin precursors, presented by the nonpolymorphic MHC class I-like molecule MR1. Here we show that human MAIT cells are remarkably oligoclonal in both the blood and liver, display high inter-individual homology and exhibit a restricted length CDR3b domain of the TCRVb chain. We extend this analysis to a second sub-population of MAIT cells expressing a semi-invariant TCR conserved between individuals. Similar to 'conventional' MAIT cells, these lymphocytes react to riboflavin-synthesizing microbes in an MR1-restricted manner and infiltrate solid tissues. Both MAIT cell types release Th0, Th1 and Th2 cytokines, and sCD40L in response to bacterial infection, show cytotoxic capacity against infected cells and promote killing of intracellular bacteria, thus suggesting important protective and immunoregulatory functions of these lymphocytes.

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Section: Cdr3b Length Distribution Differs In Mait and Non-mait Cellsmentioning

confidence: 99%

Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire

et al. 2014

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“…[1][2][3] Due to the purity of the metals and the mass resolution of ICP-MS, there is effectively no "spectral overlap." Since most of the elements used are not biologically relevant, there is also little background to cause "autofluoresence."…”

Section: Discussionmentioning

confidence: 99%

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

Maecker

2012

JoVE

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In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes. [2][3][4][5] Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples. [6][7] Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal. Video LinkThe video component of this article can be found at http://www.jove.com/video/4398/ ProtocolAll cell samples for the CyTOF must be fixed and permeabilized. This enables greater entry of the iridium-containing DNA intercalator, and also prevents cell lysis during the MilliQ water wash and resuspension steps immediately before injecting into the mass cytometer.

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“…Depending on the element of interest, MIBI can achieve as low as parts-per-billion sensitivity with a dynamic range of 10 5 and resolution comparable to high-magnification light microscopy. Instead of fluorophores or enzymeconjugated reagents, biological specimens are incubated with primary antibodies coupled to stable lanthanides highly enriched for a single isotope 32 . Primary antibodies are combined in solution for simultaneous incubation with the specimen.…”

Section: Multiplexed Ion Beam Imagingmentioning

confidence: 99%

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

Parra¹

2017

J Cancer Treat & Diagnosis

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Multiplexed immunofluorescence (IF) methods to detect simultaneously different molecules are revolutionizing immunohistochemistry (IHC) in the last years. These new technologies can be valuable for tumor examination in formalin-fixed paraffin-embedded (FFPE) specimens, and for improved new treatment discoveries and translational cancer studies. The aim of this minireview is to highlight the recent methodologies that using multiplexed IF to study simultaneous proteins identification in FFPE tumor tissues to clinical research and potential translational analysis. Multiplexed IF methods, which permit the identification of up to 4 proteins at the same time, have been increased in the last years the abilities of study cells by cells and their spatial distribution in several tumor tissues. Although, most of the old platforms are not more used after the powerful multiplex IHC methods are continue growing, the basis of these old methodologies have helped to improve the new technologies. Associated with image analysis software's these technologies can be improved to performance high throughput assay to study these specimens. Each multiplexed IF technique, detailed herein, is associated with important advantages in cancer study as well as translational research studies.

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Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry

Cited by 1,130 publications

References 29 publications

Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire

Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

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