title: Multicolor multicycle molecular profiling with quantum dots for single-cell analysis.
abstract: 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).
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington, USA.
authors.forename: Pavel
authors.initials: P
authors.lastname: Zrazhevskiy
authors.forename: Lawrence D
authors.initials: LD
authors.lastname: True
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Gao
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D045663:Quantum Dots / Q000737:chemistry*; D059010:Single-Cell Analysis / Q000379:methods*