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Designations:

L Wnt-3A

Depositors:

R Nusse

Biosafety Level:

1

Shipped:

frozen

Medium & Serum:

See Propagation

Growth Properties:

adherent

Organism:

Mus musculus （mouse）

Morphology:

fibroblast

Source:

Tissue: subcutaneous connective tissue; areolar and adipose
Strain: C3H/An

Cellular Products:

Wnt-3A protein

Permits/Forms:

In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimay responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Age:

100 days

Gender:

male

Comments:

L-M（TK-） cells （ATCC CCL-1.3） were transfected with a Wnt-3A expression vector and stable clones were selected in medium containing G418.
The Wnt-3A gene encodes a secreted glyoprotein with a variety of signaling effects. Wnt genes control many of the patterning and growth events during embryonic development.
The cells secrete biologically active Wnt-3A protein. They are presently the best source for production of Wnt-3A conditioned medium.
Since the conditioned medium contains other factors besides the Wnt-3A protein, it is necessary to control any experiments involving the Wnt-3A conditioned medium with control conditioned medium from the parental cell line （ATCC CRL-2648）.

Propagation:

ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: 0.4 mg/ml G-418; fetal bovine serum to a final concentration of 10%.
Atmosphere: air, 95%; carbon dioxide （CO2）, 5%
Temperature: 37.0°C

Subculturing:

Protocol:

Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% （w/v） Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed （usually within 5 to 15 minutes）.
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.

Protocol for Wnt-3A conditioned medium:
1. Split the cells 1:10 in 10 ml culture medium （ without G418 if conditioned medium is to be used with a cell line sensitive to G418） in 10 cm tissue culture dishes or T-75 flasks and let the cells grow for 4 days （approximay to confluency）.2. Take off the medium and sterile filter. This is the first batch of medium.3. Add 10 ml fresh culture medium and culture for another 3 days.4. Take off the medium and sterile filter. This is the second batch of medium. Discard the cells, because they will be overgrown.5. Mix the first batch and second batch of medium 1:1. This is the Wnt-3A conditioned medium. It is stable at 4° C and can be frozen.
Subc*tion Ratio: A subc*tion ratio of 1:4 to 1:10 is recommended
Medium Renewal: Every 2 to 3 days

Preservation:

Freeze medium: Complete growth medium supplemented with 5% （v/v） DMSO
Storage temperature: liquid nitrogen vapor phase

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上海復祥生物科技有限公司

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