rpc00008-T87-3.2 (2014.2.25; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00008

Arabidopsis thaliana T87 cell suspension cultureComponents• Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cellsuspension• 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 [Notes I, II].
• 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: JPL medium, 1 µM NAA (medium no. 5) [Materials III]
• Culture conditions: 22℃, continuous light, 120 rpm [Methods II]• Subculture: 14-day intervals [Methods I]
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: “Arabidopsis thaliana T87
cell line (rpc00008) was provided by the RIKEN BRC through the National
BioResource Project of the MEXT, Japan.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
JapanFAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

1

Introduction
Arabidopsis T87 cell line was established from a seedling of Arabidopsis thaliana (L.)
Heynh. accession Columbia (Axelos et al. 1992). The T87 cell culture is green and composed of small near-uniform aggregates of cells (Figure 1). The T87 cells are grown in aJouanneau and Péaud-Lenoël (JPL) medium supplemented with 1 µM 1-naphthaleneaceticacid (NAA). Our T87 cell culture has been maintained under the continuous light at 22℃
with rotary shaking at 120 rpm and subcultured at 14-day intervals (Figure 2).

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) JPL_A’
KNO3
CaCl2 ·2H2 O
MgSO4 ·7H2 O
KH2 PO465.5 mg/mL
4.4 mg/mL
3.7 mg/mL
1.7 mg/mLB) JPL_B
H3 BO3MnSO4 ·5H2 O
ZnSO4 ·7H2 O
KI
Na2 MoO4 ·2H2 OCuSO4 ·5H2 O
CoCl2 ·6H2 O

6.2 mg/mL
24.1 mg/mL
10.6 mg/mL
0.83 mg/mL
0.25 mg/mL
0.025 mg/mL
0.025 mg/mLC) JPL_C
FeSO4 ·7H2 O
2.78 mg/mL
Na2 -EDTA
3.73 mg/mL
Heat at 80℃ for 3–4 hours for chelating FeD) JPL_D
Glycinemyo-Inositol

0.2 mg/mL
10 mg/mLE) JPL_VT
Nicotinic acid
Pyridoxine·HCl

0.5 mg/mL
0.5 mg/mL

2Thiamine·HCl

0.4 mg/mLF) JPL_P
200 mM KH2 PO4200 mM Na2 HPO4H2 O

19.5 mL
30.5 mL
50 mLG) NAA (1 mM)
NAA·K
0.224 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
H) SucroseI) Casein hydrolysate, vitamin free
Casamino acids vitamin assay, Difco (#228820)
J) KOH (1 N)
K) HCl (1 N)
Glassware and equipment(All are sterilized by autoclaving at 121℃ for 20 min)A) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foilB) Pipette (10 mL; large tip opening) and a bulb
C) Stainless sieve (diameter, 5 cm; pore size, 1 mm) set on a tall beaker (200 mL),
capped with two layers of aluminum foil
Preparation of JPL medium (medium no. 5)
1. Prepare three solutions as follows.
A) JPL mineral solution (1 L)
JPL_A’
37.5 mL
JPL_B
0.375 mL
JPL_C
2.5 mL
Adjust the pH to 5.7 with KOH (1 N)
B) JPL organic solution (100 mL)
JPL_D

10 mL

3

JPL_VT
1 mL
Casein hydrolysate
0.1 g
Adjust the pH to 5.7 with HCl (1 N)
C) JPL sucrose solution (100 mL)
JPL_P
NAA (1 mM)
Sucrose

1 mL
1 mL
15 g

2. Autoclave the ﬂask at 121℃ for 20 min.
3. Add 800 mL of JPL mineral solution, 100 mL of JPL organic solution, and 100 mLof JPL sucrose solution asceptically.
4. Pour 80 mL of the medium into a sterile 300-mL ﬂask.

Methods
1. Filter a 14-day-old cell suspension through a stainless sieve (Figure 3).
2. Agitate the ﬁltrate well and transfer 2–3 mL of cell suspension to 80 mL of fresh
JPL medium with a pipette.
3. Incubate cell cultures on a rotary shaker at 120 rpm under the continuous light condition (photosynthetic photon ﬂux density 40–42 µmol m−2 s−1 ) at 22℃.

Notes
• For domestic customers: We send T87 cell suspension in a 50-mL disposable conicalcentrifuge tube. The cells should be transferred to fresh JPL medium immediately
after arrival.
• For overseas customers: We send T87 cells placed on semi-solid JPL medium
in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred tofresh JPL medium immediately after arrival. Collect the cells from the semi-solid
medium with a spatula and transfer them to Erlenmeyer ﬂasks containing fresh liquidmedium.
• In order to maintain T87 cell suspension cultures stably, it is essential to transfer anadequate amount of cells to fresh JPL medium in every subculture. The amount ofcells may vary from one lab to another, because proliferation of T87 cells is aﬀectedby culture conditions, such as a room temperature, rotation speed of a rotary shaker,
and aeration condition of the culture. T87-cell clumps occationally develop into4

large aggregates, which causes a descrease in the amount of cells passed through a1-mm sieve.
• 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; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).
• T87 cells may be maintained in Gamborg’s B5 medium (Yamada et al. 2004) or
Murashige and Skoog medium (Cunillera et al. 2000) other than JPL medium.

References
Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient
gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension
cultures. Plant Physiology and Biochemistry 30: 123–128.
Cunillera N, Arró M, Forés O, David Manzano, Ferrer A (2000) Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolicholbiosynthesis. FEBS Letters 477: 170–174. DOI: 10.1016/S0014-5793(00)01798-1
Yamada H, Koizumi N, Nakamichi N, Kiba T, Yamashino T, Mizuno T (2004) Rapid response of Arabidopsis T87 cultured cells to cytokinin through His-to-Asp phosphorelay signal transduction. Bioscience, Biotechnology, and Biochemistry 68: 1966–1976. DOI: 10.1271/bbb.68.19665Figure 1. Arabidopsis thaliana T87 cell suspension culture
A: Two-week-old cell suspension culture.
B: Cell clumps. The size of cell clumps
are varied, but most of them are below
1 mm. Scale bar = 1 mm
C: Morphology of T87 cells. T87 cells
were observed by using diﬀerential interference contrast microscopy. Thecells contain many chloroplasts. Scalebar = 50 µm

6Figure 2. Growth proﬁle of T87 cells

7Figure 3. Procedure for subculturing T87 cells
A: Two-week-old cell suspension culture.
B: Pass the culture through a stainless sieve (poresize, 1 mm).
C: Transfer 2 mL of the ﬁltrate to 80 mL of fresh
JPL medium.

8Appendix A: Formulation of culture medium
Table A.1. Jouanneau and Péaud-Lenoël medium(medium no. 5)
ChemicalConcentration (mg/L)

KNO3

1965

CaCl2 ·2H2 O

132

MgSO4 ·7H2 O

111

KH2 PO456.308

Na2 HPO48.66

H3 BO3

1.86

MnSO4 ·5H2 O

7.23

ZnSO4 ·7H2 O

3.18

KI

0.249

Na2 MoO4 ·2H2 O

0.075

CuSO4 ·5H2 O

0.0075

CoCl2 ·6H2 O

0.0075

FeSO4 ·7H2 O

5.56

Na2 -EDTA

7.46

Nicotinic acid

0.5

Pyridoxine·HCl0.5

Thiamine·HCl0.4

Glycine2

myo-Inositol

100

Casein hydrolysate, vitamin free100

Sucrose

15000

NAA·K

0.2249