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Vorobjev I.A.,Moscow State University | Rafalovskaya-Orlovskaya E.P.,Russian Hematology Scientific Center | Gladkih A.A.,Russian Hematology Scientific Center | Potashnikova D.M.,Russian Hematology Scientific Center | And 2 more authors.
Tsitologiya | Year: 2011

Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.


Vorobjev I.A.,Moscow State University | Rafalovskaya-Orlovskaya E.P.,Hematology Scientific Center | Gladkih A.A.,Hematology Scientific Center | Potashnikova D.M.,Hematology Scientific Center | And 2 more authors.
Cell and Tissue Biology | Year: 2011

Quantum dots nanocrystals (Qdots or QDs), consisting of a CdSe core with a ZnS shell, are a novel class of fluorochromes with significant advantages over traditional organic fluorochromes and fluorescent proteins. QDs have a large extinction coefficient, high photostability, wide absorption and narrow emission spectra, and large Stokes shifts. These features make them desirable for both microscopy and flow cytometry. Applications of QD-conjugates with antibodies, streptavidin, and DNA or RNA probes have made it possible to produce highly stable multicolor specimens useful for scientific and diagnostic purposes. The current review describes the achievements in preparation of multicolor specimens based on QD-conjugates for microscopy and flow cytometry and outlines the requirements for microscope and flow cytometer reengineering for successful analysis of these specimens. However, despite considerable progress, two of the obstacles that preclude wider use of QDs include some of their chemical properties and the large size of QD-conjugates. Difficulties in the application of QDs are similar whether commercial or custom-made conjugates are used. © 2011 Pleiades Publishing, Ltd.


Guan R.,Harvard University | Guan R.,Immune Disease Institute and Program in Cellular and Molecular Medicine at Childrens Hospital | Han D.,Harvard University | Harrison S.C.,Harvard University | And 3 more authors.
Structure | Year: 2010

Auxilin, a J-domain containing protein, recruits the Hsc70 uncoating ATPase to newly budded clathrin-coated vesicles. The timing of auxilin arrival determines that uncoating will commence only after the clathrin lattice has fully assembled and after membrane fission is complete. Auxilin has a region resembling PTEN, a PI3P phosphatase. We have determined the crystal structure of this region of bovine auxilin 1; it indeed resembles PTEN closely. A change in the structure of the P loop accounts for the lack of phosphatase activity. Inclusion of phosphatidylinositol phosphates substantially enhances liposome binding by wild-type auxilin, but not by various mutants bearing changes in loops of the C2 domain. Nearly all these mutations also prevent recruitment of auxilin to newly budded coated vesicles. We propose a specific geometry for auxilin association with a membrane bilayer and discuss implications of this model for the mechanism by which auxilin detects separation of a vesicle from its parent membrane. © 2010 Elsevier Ltd.

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