Multicolor multicycle molecular profiling with quantum dots for single-cell analysis
| Autor: | Pavel Zrazhevskiy, Xiaohu Gao, Lawrence D. True |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Mammals
Materials science medicine.diagnostic_test Fluorescent Antibody Technique Immunofluorescence Fluorescence Molecular biology Article General Biochemistry Genetics and Molecular Biology Staining Biological specimen Single-cell analysis Quantum dot Quantum Dots Fluorescence microscope Biophysics medicine Animals Humans Single-Cell Analysis Molecular imaging Cells Cultured HeLa Cells |
| Zdroj: | Nature Protocols. 8:1852-1869 |
| ISSN: | 1750-2799 1754-2189 |
| DOI: | 10.1038/nprot.2013.112 |
| Popis: | Here we present a detailed protocol for molecular profiling of individual cultured mammalian cells using multicolor multicycle immunofluorescence with quantum dot probes. It includes instructions for cell culture growth and processing (2 h + 48-72 h for cell growth), preparation and characterization of universal quantum dot probes (4.5 h + overnight incubation), cyclic cell staining (∼4.5 h per cycle) and image analysis (varies by application). The use of quantum dot fluorescent probes enables highly multiplexed, robust quantitative molecular imaging with a conventional fluorescence microscopy setup, whereas the probe preparation methodology, using a self-assembly between protein A-decorated universal quantum dots and intact primary antibodies, offers a fast, simple and purification-free route for an on-demand preparation of antibody-functionalized quantum dot libraries. As a result, this protocol can be used by biomedical researchers for a variety of cell staining applications, and, with further optimization, for staining of other biological specimens (e.g., clinical tissue sections). |
| Databáze: | OpenAIRE |
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