rpc00087: Bruguiera sexangula BsLs 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: AA medium, 0.02 µM 2,4-D, 2 µM 4-CPPU, pH 6.2 (medium no. 57)

    • Culture conditions: 27°C, dark, 130 rpm

    • Subculture: 21-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: “Bruguiera sexangula BsLs cell line (rpc00087) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

BsLs cell line was established from a leaf of a mangrove species Bruguiera sexangula (Lour.) Poir. (Kura-Hotta et al. 2001 [1]). The BsLs suspension cells have high salt tolerance. The BsLs cells are grown in a Amino Acid (AA) medium supplemented with 0.02 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 µM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU), pH 6.2. Our BsLs cell culture has been maintained in the dark at 27°C with rotary shaking at 130 rpm and subcultured at 21-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • Sucrose

  • CaCl2 (0.6 M)

    Chemical

    Concentration (mg/mL)

    CaCl2·2H2O

    88

  • AA_macro

    Chemical

    Concentration (mg/mL)

    KCl

    149

    MgSO4·7H2O

    37

    KH2PO4

    17

  • MS_micro_1

    Chemical

    Concentration (mg/mL)

    H3BO3

    0.62

    MnSO4·5H2O

    2.41

    ZnSO4·7H2O

    0.86

    KI

    0.083

    Na2MoO4·2H2O

    0.025

    CuSO4·5H2O

    0.0025

    CoCl2·6H2O

    0.0025

  • Fe(III)-EDTA (20 mM)

    Chemical

    Concentration (mg/mL)

    Fe(III)-EDTA

    8.422

    Ethylenediamine-N,*N*,*N*′,*N*′-tetraacetic acid, iron (III), sodium salt, trihydrate, FUJIFILM Wako Pure Chemical Corporation (#343-01241)

  • AA_5

    Chemical

    Concentration (mg/mL)

    L-Glutamine

    8.77

    L-Aspartic acid

    2.66

    L-Arginine

    2.28

    Glycine

    0.75

    Store at -20°C

  • AA_VT

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    0.05

    Pyridoxine·HCl

    0.05

    Thiamine·HCl

    0.01

    Glycine

    0.16

    Store at -20°C

  • 2,4-D (1 mM)

    Chemical

    Concentration (mg/mL)

    2,4-D sodium monohydrate

    0.261

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

  • 4-CPPU (2 mM)

    Chemical

    Concentration (mg/mL)

    4-CPPU

    0.495

    N-(2-Chloro-4-pyridyl)-N′-phenylurea, Sigma-Aldrich (C2791); Dissolve 4-CPPU in ethanol and store at -20°C

  • NaOH (1 N)

Glassware

  • Erlenmeyer flask (100 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 AA medium (medium no. 57)

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

    Chemical

    Amount

    Sucrose

    30 g

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

    Stock solution

    Volume (mL)

    CaCl2 (0.6 M)

    5

    AA_macro

    10

    MS_micro_1

    10

    Fe(III)-EDTA (20 mM)

    5

    AA_5

    100

    AA_VT

    10

    2,4-D (1 mM)

    0.02

    4-CPPU (2 mM)

    1

  3. Adjust the pH of the solution to 6.2 with NaOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 20 mL of the medium into a 100-mL flask.

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

Methods

  1. Agitate a 21-day-old culture well and transfer 2 mL of cell suspension to 20 mL of fresh AA medium with a pipette.

  2. Incubate cell cultures on a rotary shaker at 130 rpm under the dark condition at 27°C.

Notes

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

  • For overseas customers: We send BsLs cells placed on semi-solid AA medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh AA 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 BsLs cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh AA medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of BsLs cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.

  • 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