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
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. "
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.
- "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). "
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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 "
ABSTRACT: Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be 'trapped' on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.
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