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Cell cycle staging of individual cells by fluorescence microscopy

Nature Protocols volume 10, pages 334–348 (2015)Cite this article

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Abstract

Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1–2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.

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Figure 1: Overview of the protocol.
Figure 2: Cell cycle profiles derived by using high-throughput confocal fluorescence microscopy.
Figure 3: Cell cycle histograms obtained by using standard wide-field fluorescence microscopy.
Figure 4: Calculation of population of cells within distinct cell cycle phases.

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Acknowledgements

Work in the Misteli laboratory and in the High-Throughput Imaging Facility is supported by the Intramural Research Program of the US National Institutes of Health (NIH), National Cancer Institute (NCI), Center for Cancer Research. We thank S. Burke for modeling the cell cycle data, K. MacKinnon for help with flow cytometry and T. Karpova (NCI Fluorescence Imaging Facility) for help with microscopy.

Author information

Author notes

  1. Gianluca Pegoraro and Ty C Voss: These authors contributed equally to this work.

Authors and Affiliations

  1. National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA

    Vassilis Roukos, Gianluca Pegoraro, Ty C Voss & Tom Misteli

  2. High-Throughput Imaging Facility, National Cancer Institute, NIH, Bethesda, Maryland, USA

    Gianluca Pegoraro

  3. PerkinElmer Health Sciences Inc., Waltham, Maryland, USA

    Ty C Voss

Authors
  1. Vassilis Roukos
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  4. Tom Misteli
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Contributions

V.R., T.C.V. and G.P. developed the protocol; V.R wrote the manuscript; G.P edited the manuscript; and T.M. supervised the study and edited the manuscript.

Corresponding authors

Correspondence to Vassilis Roukos or Tom Misteli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimization of the imaging parameters for Option A.

A. Selection of the optimal middle, upper and lower planes in z of the field shown in Figure 2A. B. Images of sequential 1μm z stacks of nuclei stained with DAPI. The middle plane (marked by a red box) is selected as the image with the higher nuclei average intensity (Av). The upper plane (yellow box) and the lower plane (turquoise box), are selected to include cells in mitosis (white arrows) and to range the same distance from the middle plane (lower plane: z=-7μm, middle plane: z=0, upper plane: z=+7μm). C. DNA content histograms derived by image analysis on three focal plane average projections (left panel) and by analysis of only the middle plane (right panel). The inability to capture mitotic cells when the middle plane is only acquired is reflected by the lower percentage of cells within the G2/M population (right panel).

Supplementary Figure 2 Cell cycle profiles obtained using different numbers of NIH3T3 cells.

Cells were plated in 16 wells of a 384 well imaging plate and processed as described in Option A of the protocol. Histograms were plotted by pooling data from 16, 10, 8, 4, 2 or a single well (9 different fields per well) corresponding to ~7500, 4500, 3500, 1800, 900 and 500 cells, respectively.

Supplementary Figure 3 Examples of text file structures produced in Step 4Av and required for Step 4Avii.

A. The text file structure that is produced by the image analysis in Step 4Av opened in Excel file. Column F, named attribute “Z_IntegratedDAPIInt” (marked by the red arrow) shows the averaged z integrated nuclear intensity of each individual nucleus detected per well. B. An example of a “GlobalLayout.txt” table file that maps the experimental treatments to the appropriate well positions by row and column numbers is shown.

Supplementary Figure 4 Direct comparison of the cell cycle distributions derived by DNA content analysis using flow cytometry (propidium iodide staining, left side) or produced by single-cell fluorescence microscopy (DAPI staining, right side) by following this protocol.

Flow cytometry and image analysis data obtained by high-throughput (A) or wide-field microscopy (B) of control cells or cells treated with etoposide, were imported in FCS Express and modeled via the multicycle plugin. The percentage of cells within the different cell cycle phases is shown.

Supplementary information

Supplementary Figures

Supplementary Figures 1–4 (PDF 645 kb)

Supplementary Data

Scripts and example imaging data for testing purposes. (ZIP 50657 kb)

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Roukos, V., Pegoraro, G., Voss, T. et al. Cell cycle staging of individual cells by fluorescence microscopy. Nat Protoc 10, 334–348 (2015). https://doi.org/10.1038/nprot.2015.016

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