CellVue Claret, a new far-red dye, facilitates polychromatic assessment of immune cell proliferation.
ABSTRACT Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677 nm), as an alternative and/or complementary probe to PKH26 and CFSE(1) for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0-96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm-658 nm) excitation capabilities.
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ABSTRACT: There are several statistics that may be calculated to characterize a cellular proliferation experiment. By far, the most commonly-reported statistic is the percent of cells in the final culture that have divided; however, this statistic has significant limitations. Other statistics provided by software modeling provide a much richer characterization of the biological response; however, their use also comes with caveats. Here, I discuss the practical application of these statistics, including their limitations and interdependencies, using hypothetical data. The goal of this perspective is to prevent the blind reliance or overly optimistic ("panglossian") interpretation of the statistics generated by software, so that researchers and reviewers have a more-informed basis for drawing conclusions from the data.Cytometry Part A 02/2011; 79(2):95-101. · 3.73 Impact Factor
Article: Homo sapiens systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) protein mediates contact-dependent small RNA transfer and microRNA-21-driven chemoresistance.[show abstract] [hide abstract]
ABSTRACT: Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents.Journal of Biological Chemistry 12/2011; 287(8):5267-77. · 4.77 Impact Factor