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).

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Figure 1: Workflow of the M3P technology.
Figure 2: Multiplexed one-step staining of clinical tissue specimens with self-assembled QDot-SpA-antibody probes.
Figure 3: Purification of activated QDots with NAP-5 column.
Figure 4: Single-cell molecular profiling with the M3P technology.
Figure 5: Antigen preservation via proper prestaining specimen processing.

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Acknowledgements

This work was supported in part by the US National Institutes of Health (R01CA131797 to X.G.; P50CA097186 to L.D.T.), the US National Science Foundation (NSF) (0645080), US Department of Defense–Congressionally Directed Medical Research Programs (DoD-CDMRP) (W81XWH0710117), the Coulter foundation and the Department of Bioengineering at the University of Washington. X.G. thanks the NSF for a Faculty Early Career Development award (CAREER). P.Z. thanks the University of Washington Center for Nanotechnology for a University Initiatives Fund Fellowship, the NSF for a Graduate Research Fellowship (DGE-0718124) and the National Cancer Institute for a T32 Fellowship (T32CA138312). We are also grateful to R. Vessella and P. Nelson for fruitful discussions on clinical diagnostics and molecular pathology research and to J. Li, C. Probst and J. Shang for valuable comments on the manuscript.

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P.Z., L.D.T. and X.G. contributed to the experiment design and data analysis. P.Z. performed the experiments. P.Z. and X.G. wrote the paper.

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Correspondence to Xiaohu Gao.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Examination of expanded molecular target sets through multicolor multicycle staining (PDF 266 kb)

Supplementary Figure 2

Achieving QDot probe intra-nuclear access with Proteinase K digestion (PDF 305 kb)

Supplementary Figure 3

Hyperspectral imaging of cells labeled by multicolor QDot probes (PDF 317 kb)

Supplementary Figure 4

Building a reference QDot spectral library with HSI (PDF 311 kb)

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Zrazhevskiy, P., True, L. & Gao, X. Multicolor multicycle molecular profiling with quantum dots for single-cell analysis. Nat Protoc 8, 1852–1869 (2013). https://doi.org/10.1038/nprot.2013.112

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