rpc00091: Nicotiana tabacum TBY2-31/ST transgenic cell suspension culture

Note

  • Components

    • Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension

    • Overseas delivery: A 250-mL plastic Erlenmeyer flask, containing cells placed on semi-solid medium

  • Notice

    • Subculture the cells to fresh medium immediately after arrival.

    • Do not store the cell culture in a refrigerator and a freezer.

    • Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.

  • Method

    • Culture medium: mLS medium, 0.2 mg/L 2,4-D, pH 5.8 (medium no. 1)

    • Culture conditions: 27°C, dark, 110 rpm

    • Subculture: 7-day intervals

  • Citation of cell line

    When results obtained by using this cell line are published in a scientific journal, it should be cited in the following manner: “Nicotiana tabacum TBY2-31/ST cell line (rpc00091) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Tobacco TBY2-31/ST cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with Arabidopsis SYP31 and monomeric Red Fluorescent Protein (mRFP) fused with rat sialyltransferase (Ito et al. 2012 2). GFP fluorescence is observed in cis-Golgi, and mRFP fluorescence is in trans-Golgi, using a fluorescence microscope. The parent cell line BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana tabacum L. cultivar Bright Yellow 2 (Nagata et al. 1992 3). The TBY2-31/ST cells are grown in a modified Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.8. Our TBY2-31/ST cell culture has been maintained in the dark at 27°C with rotary shaking at 110 rpm and subcultured at 7-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

  • Sucrose

  • BY2_P

    Chemical

    Concentration (mg/mL)

    KH2PO4

    80

  • LS_VT_modified

    Chemical

    Concentration (mg/mL)

    Thiamine·HCl

    0.4

    myo-Inositol

    40

  • 2,4-D (0.2 mg/mL)

    Chemical

    Concentration (mg/mL)

    2,4-D sodium monohydrate

    0.236

    (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

  • KOH (1 N)

Glassware

  • Erlenmeyer flask (100 mL), capped with two layers of aluminum foil

  • Pipette (2 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min

Preparation of mLS medium (medium no. 1)

  1. Dissolve the following chemicals in approximately 800 mL of distilled water.

    Chemical

    Amount

    MS salt mix

    1 bag (1 L)

    Sucrose

    30 g

  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    BY2_P

    2.5

    LS_VT_modified

    2.5

    2,4-D (0.2 mg/mL)

    1

  3. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 30 mL of the medium into a 100-mL flask.

  5. Autoclave the flask at 121°C for 20 min.

Methods

  1. Agitate a 7-day-old culture well and transfer 0.4 mL of cell suspension to 30 mL of fresh mLS medium with a pipette.

  2. Incubate cell cultures on a rotary shaker at 110 rpm under the dark condition at 27°C.

Notes

  • For domestic customers: We send TBY2-31/ST cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh mLS medium immediately after arrival. Transfer settled cells to Erlenmeyer flasks containing fresh liquid medium with a pipette.

  • For overseas customers: We send TBY2-31/ST cells placed on semi-solid mLS medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh mLS medium immediately after arrival. Collect the cells from the semi-solid medium with a spatula and transfer them to Erlenmeyer flasks containing fresh liquid medium.

  • In order to maintain TBY2-31/ST cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh mLS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of TBY2-31/ST cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.

  • A low growth rate of the parent BY-2 cells is sometimes caused by poor aeration (Kumagai-Sano et al. 2007 1). In order to obtain good aeration of a suspension culture, a silicone sponge plug may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

Maintenance history

References

1

Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S (2007) Cell cycle synchronization of tobacco BY-2 cells. Nature Protocols 1: 2621–2627. DOI: 10.1038/nprot.2006.381

2

Ito Y, Uemura T, Shoda K, Fujimoto M, Ueda T, Nakano A (2012) cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells. Molecular Biology of the Cell 23: 3203–3214. DOI: 10.1091/mbc.E12-01-0034

3

Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. International Review of Cytology 132: 1–30. DOI: 10.1016/S0074-7696(08)62452-3