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Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry.

ABSTRACT: Semiconductor nanocrystals such as quantum dots (QDs) are a potentially powerful resource in the fields of flow cytometry and fluorescence microscopy. QD size and fluorescence characteristics offer attractive features for use in targeted delivery systems and detection by flow cytometry. While quantitative measurements of a variety of fluorescent molecules are routinely performed, fluorophores for which no calibration standards exist, such as QDs, pose a problem for quantitation in flow cytometry. Our goal was to develop a targeted nanoparticle delivery platform as well as a corresponding method to accurately and quantitatively assess the performance of this system. We synthesized surface-modified QD probes targeted to cellular surface receptors and measured the MFI of the resulting cell-probe conjugates by flow cytometry. MFI was converted to mean equivalent R-PE intensity (MEPE) using standard calibration microspheres. Known concentrations of both R-PE and QD probes were measured by fluorometry to relate R-PE and QD fluorescence. Fluorometry results were then used to translate MEPE measurements to the number of bound QD probes. The targeted probes exhibited superior binding characteristics over unmodified and untargeted particles. This binding interaction was shown to be specific and mediated by the NGR targeting peptide tethered to the QD surface. The calibration method developed to assess this system proved successful at converting raw fluorescence data to quantitative probe binding values. We demonstrate the synthesis and performance of a highly modular nanoparticle system capable of targeted binding and fluorescent imaging. The calibration method implemented to quantify the performance of this system represents a potentially powerful tool to utilize truly quantitative flow cytometry measurements with an array of fluorescent molecules, including QDs.

SUBMITTER: Smith RA

PROVIDER: S-EPMC5568117 | biostudies-other | 2009 May

REPOSITORIES: biostudies-other

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Project description:Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus facilitating progress in quantitative studies of lymphocyte responses.

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Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry.

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