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Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle

Nature Protocols volume 8, pages 602–626 (2013)Cite this article

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Abstract

This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle–related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type–restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.

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Figure 1: Isolation of nuclei for use in traditional and improved cell cycle studies.
Figure 2: Nucleocytoplasmic protein tracking throughout the cell cycle via traditional immunoblotting.
Figure 3: Refined approach of flow cytometric data analysis.
Figure 4: Flow cytometric characterization of isolated nuclei.
Figure 5: Monitoring protein expression during the cell cycle via flow cytometry.
Figure 6: Monitoring nucleocytoplasmic protein expression via flow cytometry.
Figure 7: Nucleocytoplasmic protein tracking throughout the cell cycle via improved flow cytometry.

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Acknowledgements

The clonal human amniotic fluid stem cell line Q1 was kindly provided by A. Atala (Wake Forest University School of Medicine). CDKN1B (p27), TSC2 (tuberin) and TOP2B (topoisomerase IIβ) wild-type and knockout cells were obtained from J.M. Roberts (Fred Hutchinson Cancer Research Center), D.J. Kwiatkowski (Brigham and Women's Hospital) and N. Adachi (Yokohama City University), respectively. We thank all members of our laboratory for helpful comments and discussion.

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  1. Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria

    Margit Rosner, Katharina Schipany & Markus Hengstschläger

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  1. Margit Rosner
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Contributions

M.R. and K.S. designed and performed experiments, analyzed data and wrote the article. M.H. conceived the method, designed experiments, analyzed data and wrote the article.

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Correspondence to Markus Hengstschläger.

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Supplementary information

Supplementary Figure 1

Optimized fixation conditions for the combined analysis of DNA and protein on the flow cytometer. (PDF 554 kb)

Supplementary Figure 2

Evaluation of FSC (forward scatter) analyses to study cell cycle-associated changes in cell size. (PDF 460 kb)

Supplementary Figure 3

Approach and quality control of FSC (forward scatter) dissection. (PDF 788 kb)

Supplementary Figure 4

Example of a complete FSC(G1)/DNA(S)/FSC(G2/M) dissection. (PDF 421 kb)

Supplementary Figure 5

Evaluation of antibody specificity. (PDF 716 kb)

Supplementary Figure 6

Evaluation of antibody sensitivity and the comparative analysis of flow cytometry and immunoblotting with regard to the quantifiability of obtained results. (PDF 717 kb)

Supplementary Figure 7

Flow cytometric Oct-4 analyses in Q1 human amniotic fluid stem (hAFS) cells and human teratocarcinoma cells. (PDF 305 kb)

Supplementary Figure 8

Cell cycle regulation of histone H3 and α-tubulin in Q1 human amniotic fluid stem cells. (PDF 369 kb)

Supplementary Figure 9

Expression and cell cycle regulation of α-tubulin in TSC2+/+versus TSC2−/−MEFs. (PDF 298 kb)

Supplementary Figure 10

Ratios of nuclear to whole cell MFIs (FL1-H median fluorescence intensities) for cytofluorometrically analyzed nuclear, cytoplasmic and nucleocytoplasmic proteins. (PDF 270 kb)

Supplementary Figure 11

Depiction of fold changes in the cell cycle regulation of cytoplasmic versus nuclear importin α1 and mTOR. (PDF 283 kb)

Supplementary Figure 12

Cell cycle regulation of αtubulin in BxPC-3 pancreas carcinoma cells. (PDF 381 kb)

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Rosner, M., Schipany, K. & Hengstschläger, M. Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle. Nat Protoc 8, 602–626 (2013). https://doi.org/10.1038/nprot.2013.011

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  • DOI: https://doi.org/10.1038/nprot.2013.011

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