Multicolor multicycle molecular profiling with quantum dots for single-cell analysis


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.


  1. 1

    de Souza, N. Single-cell methods. Nat. Methods 9, 35 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Chaurand, P., Sanders, M.E., Jensen, R.A. & Caprioli, R.M. Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections. Am. J. Pathol. 165, 1057–1068 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Liotta, L. & Petricoin, E. Molecular profiling of human cancer. Nat. Rev. Genet. 1, 48–56 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Gorg, A., Weiss, W. & Dunn, M.J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685 (2004).

    Article  Google Scholar 

  5. 5

    Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Schwamborn, K. & Caprioli, R.M. Molecular imaging by mass spectrometry–looking beyond classical histology. Nat. Rev. Cancer 10, 639–646 (2010).

    CAS  Article  Google Scholar 

  7. 7

    Wollscheid, B. et al. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat. Biotechnol. 27, 378–386 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Caldwell, R.L. & Caprioli, R.M. Tissue profiling by mass spectrometry–A review of methodology and applications. Mol. Cell. Proteomics 4, 394–401 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Chattopadhyay, P.K. et al. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat. Med. 12, 972–977 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Ma, C. et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat. Med. 17, 738–743 (2011).

    CAS  Article  Google Scholar 

  12. 12

    Salehi-Reyhani, A. et al. A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection. Lab Chip 11, 1256–1261 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Umemura, S. & Osamura, R.Y. Utility of immunohistochemistry in breast cancer practice. Breast Cancer 11, 334–338 (2004).

    Article  Google Scholar 

  14. 14

    True, L.D. Quantitative immunohistochemistry: a new tool for surgical pathology? Am. J. Clin. Pathol. 90, 324–325 (1988).

    CAS  Article  Google Scholar 

  15. 15

    Micheva, K.D., Busse, B., Weiler, N.C., O'Rourke, N. & Smith, S.J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Schubert, W. et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24, 1270–1278 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Medintz, I.L. & Mattoussi, H. Quantum dot-based resonance energy transfer and its growing application in biology. Phys. Chem. Chem. Phys. 11, 17–45 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Medintz, I.L., Mattoussi, H. & Clapp, A.R. Potential clinical applications of quantum dots. Int. J. Nanomedicine 3, 151–167 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Misra, R.D. Quantum dots for tumor-targeted drug delivery and cell imaging. Nanomedicine 3, 271–274 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R. & Nann, T. Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 5, 763–775 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Smith, A.M., Duan, H., Mohs, A.M. & Nie, S. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60, 1226–1240 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Tholouli, E. et al. Quantum dots light up pathology. J. Pathol. 216, 275–285 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Xing, Y. & Rao, J. Quantum dot bioconjugates for in vitro diagnostics and in vivo imaging. Cancer Biomark. 4, 307–319 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Zrazhevskiy, P. & Gao, X. Multifunctional quantum dots for personalized medicine. Nano Today 4, 414–428 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Zrazhevskiy, P., Sena, M. & Gao, X.H. Designing multifunctional quantum dots for bioimaging, detection, and drug delivery. Chem. Soc. Rev. 39, 4326–4354 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Yezhelyev, M.V. et al. In situ molecular profiling of breast cancer biomarkers with multicolor quantum dots. Adv. Mater. 19, 3146–3151 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Matsuno, A., Itoh, J., Takekoshi, S., Nagashima, T. & Osamura, R.Y. Three-dimensional imaging of the intracellular localization of growth hormone and prolactin and their mRNA using nanocrystal (Quantum dot) and confocal laser scanning microscopy techniques. J. Histochem. Cytochem. 53, 833–838 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Shi, C. et al. Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells. Eur. J. Histochem. 52, 127–134 (2008).

    CAS  Article  Google Scholar 

  29. 29

    Liu, J., Lau, S.K., Varma, V.A., Kairdolf, B.A. & Nie, S. Multiplexed detection and characterization of rare tumor cells in Hodgkin's lymphoma with multicolor quantum dots. Anal. Chem. 82, 6237–6243 (2010).

    CAS  Article  Google Scholar 

  30. 30

    Liu, J. et al. Molecular mapping of tumor heterogeneity on clinical tissue specimens with multiplexed quantum dots. ACS Nano 4, 2755–2765 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Chen, C. et al. Quantum-dot–based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneity. Nanotechnology 21, 095101 (2010).

    Article  Google Scholar 

  32. 32

    Sweeney, E. et al. Quantitative multiplexed quantum dot immunohistochemistry. Biochem. Biophys. Res. Commun. 374, 181–186 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Fountaine, T.J., Wincovitch, S.M., Geho, D.H., Garfield, S.H. & Pittaluga, S. Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots. Mod. Pathol. 19, 1181–1191 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Huang, D.H. et al. Comparison and optimization of multiplexed quantum dot–based immunohistofluorescence. Nano Res. 3, 61–68 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Xing, Y. et al. Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat. Protoc. 2, 1152–1165 (2007).

    CAS  Article  Google Scholar 

  36. 36

    Zrazhevskiy, P. & Gao, X. Quantum dot imaging platform for single-cell molecular profiling. Nat. Commun. 4, 1619 (2013).

    Article  Google Scholar 

  37. 37

    Denysenko, T. et al. Glioblastoma cancer stem cells: heterogeneity, microenvironment and related therapeutic strategies. Cell Biochem. Funct. 28, 343–351 (2010).

    CAS  Article  Google Scholar 

  38. 38

    Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A. & Nolan, G.P. Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Ciftlik, A.T., Lehr, H.-A. & Gijs, M.A.M. Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and significantly reduces ambiguous (2+) read-outs. Proc. Natl. Acad. Sci. USA 110, 5363–5368 (2013).

    CAS  Article  Google Scholar 

  40. 40

    Chang, K.H. et al. Novel 16-minute technique for evaluating melanoma resection margins during Mohs surgery. J. Am. Acad. Dermatol. 64, 107–112 (2011).

    Article  Google Scholar 

  41. 41

    Goldman, E.R. et al. Avidin: a natural bridge for quantum dot-antibody conjugates. J. Am. Chem. Soc. 124, 6378–6382 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Jaiswal, J.K., Mattoussi, H., Mauro, J.M. & Simon, S.M. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 21, 47–51 (2003).

    CAS  Article  Google Scholar 

  43. 43

    Jin, T. et al. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells. Mol. BioSyst. 6, 2325–2331 (2010).

    CAS  Article  Google Scholar 

  44. 44

    Mattoussi, H. et al. Self-assembly of CdSe-ZnS quantum dot bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 122, 12142–12150 (2000).

    CAS  Article  Google Scholar 

  45. 45

    Levenson, R.M. Spectral imaging and pathology: seeing more. Lab Med. 35, 244–251 (2004).

    Article  Google Scholar 

  46. 46

    True, L.D. & Gao, X. Quantum dots for molecular pathology: their time has arrived. J. Mol. Diagn. 9, 7–11 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Byers, R.J. et al. Semiautomated multiplexed quantum dot-based in situ hybridization and spectral deconvolution. J. Mol. Diagnostics 9, 20–29 (2007).

    CAS  Article  Google Scholar 

  48. 48

    Ghazani, A.A. et al. High-throughput quantification of protein expression of cancer antigens in tissue microarray using quantum dot nanocrystals. Nano Lett. 6, 2881–2886 (2006).

    CAS  Article  Google Scholar 

  49. 49

    Toth, Z.E. & Mezey, E. Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J. Histochem. Cytochem. 55, 545–554 (2007).

    CAS  Article  Google Scholar 

  50. 50

    Glass, G., Papin, J.A. & Mandell, J.W. SIMPLE: a sequential immunoperoxidase labeling and erasing method. J. Histochem. Cytochem. 57, 899–905 (2009).

    CAS  Article  Google Scholar 

  51. 51

    Pirici, D. et al. Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype. J. Histochem. Cytochem. 57, 567–575 (2009).

    CAS  Article  Google Scholar 

  52. 52

    Wahlby, C., Erlandsson, F., Bengtsson, E. & Zetterberg, A. Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry 47, 32–41 (2002).

    CAS  Article  Google Scholar 

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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.

Author information




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.

Corresponding author

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).

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