Chattopadhyay, PK, Price, DA, Harper, TF, Betts, MR, Yu, J, Gostick, E et al.. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12: 972-977

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA.
Nature Medicine (Impact Factor: 27.36). 09/2006; 12(8):972-7. DOI: 10.1038/nm1371
Source: PubMed


Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

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    • "Higher spatial resolution is provided by multimodality laser scanning confocal microscopy (Peng et al., 2012) or " super-resolution " techniques (Huang et al., 2008; Enderlein, 2012; Liu et al., 2012). Analyzing a large population of cells, however, has demanded other approaches such as automated cell analysis by scanning cytometry (Kamentsky and Kamentsky, 1991; Bae et al., 2012), flow cytometry (Shapiro, 2003; Perfetto et al., 2004; Chattopadhyay et al., 2006; Grégori et al., 2011; Robinson et al., 2012), and automated imaging flow cytometry (Mirabelli et al., 2012). Most fluorescence probes, including organic dyes, fluorescent proteins, and nanoparticles, are sensitive enough to reveal sub-cellular information. "
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    ABSTRACT: The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes. Three detailed, step-by-step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time-gated luminescence. Curr. Protoc. Cytom. 67:2.22.1-2.22.36. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2014; 67:2.22.1-2.22.36. DOI:10.1002/0471142956.cy0222s67
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    • "Tetramer production: In the past, only FITC-, PE-, APC-tetramers were available, which limited panel design because many novel or dimly staining antibodies are only found on these fluorochromes. QDs with SA groups can be used to produce pMHCI multimers (commonly called 'tetramers') (Chattopadhyay et al, 2006), displaying higher valency than PE or APC and, thus, allowing brighter signals and better staining resolution (Chattopadhyay et al, 2008). "
    Flow Cytometry – Recent Perspectives, Edited by Schmid I, 01/2012: chapter Applications of Quantum Dots in Flow Cytometry; InTech Publisher., ISBN: 979-953-307-355-1
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    • "For intracellular staining, mAbs included anti-CD3 Qdot 585 (custom) and anti-IFN-γ Alexa-700 (BD Pharmingen, San Diego, California). Custom conjugations to quantum dot (Qdot) nanocrystals were performed in our laboratory as described previously (Chattopadhyay et al., 2006) with reagents purchased from Invitrogen. Peptide/MHC class I tetramers were produced as described previously.(Price "
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