rpc00040: Nicotiana tabacum GF11 transgenic cell suspension culture¶
- Domestic delivery: Two 50-mL tubes, containing 25 mL of cell suspension
- Overseas delivery: Two 250-mL flasks, containing cells placed on semi-solid medium
- 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.
- Culture medium: mLS medium, 0.2 mg/L 2,4-D, pH 5.8 (medium no. 1)
- Culture conditions: 27°C, dark , 130 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 GF11 cell line (rpc00040) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”
Tobacco GF11 cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with Arabidopsis fimbrin second actin-binding domain (ABD2) (Sano et al. 2005 ). GFP fluorescence is observed in actin microfilaments by 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 ). The GF11 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 GF11 cell culture has been maintained in the dark at 27°C with rotary shaking at 130 rpm and subcultured at 7-day intervals.
Chemicals and stock solutions¶
(All stock solutions are stored at 4°C)
MS salt mix
Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (#392-00591)
Chemical Concentration (mg/mL) KH2PO4 80
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)
- Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
- Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min
Preparation of mLS medium (medium no. 1)¶
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical Amount MS salt mix 1 bag (1 L) Sucrose 30 g
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
Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.
Pour 95 mL of the medium into a 300-mL flask.
Autoclave the flask at 121°C for 20 min.
- Agitate a 7-day-old culture well and transfer 3–3.2 mL of cell suspension to 95 mL of fresh mLS medium with a pipette.
- Incubate cell cultures on a rotary shaker at 130 rpm under the dark condition at 27°C.
- For domestic customers: We send GF11 cell suspension in 50-mL disposable tubes. 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 GF11 cells placed on semi-solid mLS medium in 250-mL disposable flasks. 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 GF11 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 GF11 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 ). In order to obtain good aeration of a suspension culture, a silicone cap may be used instead of the aluminum foil cap.
|||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|
|||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|
|||Sano T, Higaki T, Oda Y, Hayashi T, Hasezawa S (2005) Appearance of actin microfilament ‘twin peaks’ in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin. Plant Journal 44: 595–605. DOI: 10.1111/j.1365-313X.2005.02558.x|