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Cell cycle synchronization of tobacco BY-2 cells

Nature Protocols volume 1, pages 2621–2627 (2006)Cite this article

Abstract

Synchronization is a powerful technique for understanding cell cycle events. Here, we describe the procedure for synchronizing tobacco bright yellow 2 (BY-2) cell line, with which an exceptionally high level of synchrony can be achieved. It basically relies on an “arrest-and-release” strategy using aphidicolin, an inhibitor of DNA replication, and propyzamide, a plant-microtubule disruptant. In a single-step process using aphidicolin alone, a cell population with about 70% of the cells at mitosis can be achieved, whereas by a two-step method using the two inhibitors sequentially, the level of synchrony can reach over 90%. The method of choice depends not only on the peak mitotic cell proportion but also on the cell cycle stage that is targeted for analysis. Both procedures take about 1.5 days, and cell cycle progression can be observed from the S phase to the next G1 phase at about 12 h after a 24 h-period treatment with aphidicolin.

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Figure 1: A bright-field image of tobacco BY-2 cells at 6 h after aphidicolin release.
Figure 2: Brief illustration of washing equipment.
Figure 3: Fluorescence microscopic images of nuclei and chromosomes of BY-2 cells at various stages of the cell cycle.
Figure 4: Anticipated results of BY-2 synchronization.

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Authors and Affiliations

  1. Department of Science Education, Faculty of Education, Gunma University, Aramaki-cho 4-2, Maebashi, 371-8510, Gunma, Japan

    Fumi Kumagai-Sano

  2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, 277-8562, Chiba, Japan

    Tomomi Hayashi, Toshio Sano & Seiichiro Hasezawa

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  1. Fumi Kumagai-Sano
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Correspondence to Fumi Kumagai-Sano.

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Kumagai-Sano, F., Hayashi, T., Sano, T. et al. Cell cycle synchronization of tobacco BY-2 cells. Nat Protoc 1, 2621–2627 (2006). https://doi.org/10.1038/nprot.2006.381

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