Cell Line



BCRC Number: 60469
Name: ES-D3 GL
Type: mouse embryonic stem cell
Species: Mus musculus (mouse)
Strain Ethnicity: 129/Sv+c/+p
Age Stage: embryo, blastocysts
Gender: unknown
Tissue: embryo; blastocysts; germline competent

Morphology: embryonic stem cell
Split Ratio: 1:3 to 1:4 every 2 to 3 days
Mycoplasma Test: negative

Incubation Temp(°C): 37°C
Incubation Condition: 5% CO2
Culture Medium: Dulbecco's modified Eagle's medium with 4 mM L-glutamine and 4.5 g/L glucose, supplemented with 10% fetal bovine serum, 15 mM HEPES, 1.2 g/L sodium bicarbonate, 200 U/ml penicillin-G, 0.2 mg/ml streptomycin, 0.025 mg/ml ampicillin, and add fresh from concentrated(1000x)stocks: 0.1mM 2-mercaptoethanol 1,000U/ml,10ng/ml of LIF.
Growth Property: adherent; NOTE: A feeder layer must be used to grow these cells. Feeder cells: These cells were grown on mitomycin C treated STO SNL-2 feeder cells (STO cells constitutively expressing the human LIF gene) and supplemented with 10 ng/ml of mouse LIF. STO SNL2 feeder cells can be replaced by other mouse embryo fibroblast cells.
SubCulture Procedure: 02% trypsin-1 mM EDTA; NOTE: Once the clumps are dislodged from the feeder cells, collect the cells in 5 to 7 ml of fresh culture medium, centrifuge, and resuspend the cells in fresh medium. Dispense onto fresh feeder layer cultures.
Freezed Medium: 40% culture medium + 50% fetal bovine serum + 10% DMSO
Seeding Density: 2 x 106 to 4 x 106 cells/T25; NOTE: If the spheres are touching each other, the culture is overgrown. Do not let the ES cell clumps fuse.
Medium Renewal: every 1 to 2 days or when culture medium becomes acid

Description: 'The clonal embryonic stem cell line ES-D3 was derived from a strain of 129/Sv+C/+P mouse blastocyst. The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium. They can be maintained in the undifferentiated state by frequent subculture (every 2 to 3 days) on confluent feeder layers of irradiated (12000 rads) or mitomycin C treated (0.01 mg/ml for 2 hours) primary mouse embryonic fibroblasts or STO cells. Leukemia Inhibitory Factor (LIF) may also be added to ES-D3 cells cultured on feeder layers for additional support of the undifferentiated state. This cell line is a derivative of the ES-D3 cell line originally established by Tom Doetschman.

Image / Description:   200倍視野下 上層的細胞為ES-D3 GL 下層的細胞feeder cells
 

Biosafety Level: 1
Reference: J. Embryol. Exp. Morphol. 87: 27-45, 1985; Nature 336: 684-687, 1988; Proc. Natl. Acad. Sci. USA 83: 9065-9069, 1986; Proc. Natl. Acad. Sci. USA 85: 8583-8587, 1988
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