Development of high-throughput analytical system for intracellular molecules with quantum dots

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Fluorescent semiconductor quantum dots (QDs) are a novel class of fluorescent probe with optical properties that differ from those of conventional fluorochromes. QDs emit bright fluorescence at visible wavelengths, depending on size of the particle, and in addition this bright fluorescence is resistant to photobleaching. In this project, we aimed to demonstrate that QDs are useful to quantify intracellular materials and applied to cytometry. First, QDs were substituted for organic fluorochromes in immunohistochemistry. Some intracellular/nuclear antigens were detected by secondary antibodies labeled with QDs, but some antigens were not. This may be caused by the difference of physiochemical property between two types of fluoroprobes and we tried various fixations and dying procedure to find optimal condition for immunohistochemistry with QDs. For example, samples needed treatment with Triton X-100 and microwave exposure in sodium citrate buffer (pH6) for the purpose of the detection of … More intranuclear proliferating cell nuclear antigen (PCNA). We plan to determine the optimal methods for various antigens. We verified whether QDs bind to their targets stoichiometrically as well as classical fluorochromes. We labeled PCNA in nuclei of PC-14 cells immunocytochemically with QDs and Alexa 488 concomitantly and measured the level of intranuclear PCNA expression during cell cycle with laser scanning cytometry. At the same time, nuclear DNA stained with propidium iodide (PI) was measured. The bivariate DNA/PCNA content cytograms for QDs and Alexa 488 fluorescence were dome-shaped and very similar to each other. These indicated that immunocytochemistry with QDs under optimal condition provided the data equivalent to those with organic fluorescence dyes and QDs could be useful fluorochromes in cytometry. However, we didn't always succeed to detect fluorescence intensity by LSC or flow cytometer and we need further examination for immunocytochemical methods and optical condition in cytometry. Less