L Wnt-3A (ATCC® CRL-2647)

Organism: Mus musculus, mouse  /  Tissue: subcutaneous connective tissue; areolar and adipose  / 

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Organism Mus musculus, mouse
Tissue subcutaneous connective tissue; areolar and adipose
Product Format frozen
Morphology fibroblast
Culture Properties adherent
Biosafety Level 1
Age 100 days
Gender male
Strain C3H/An
Applications
This cell line is presently the best source for production of Wnt-3A conditioned medium.
Storage Conditions liquid nitrogen vapor phase
Derivation
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.
Clinical Data
male
Genes Expressed
Wnt-3A
Cellular Products
Wnt-3A protein
Comments

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).
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%.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium.
  2. 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.
  3. 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.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by pipetting gently.
  5. Add appropriate aliquots of cell suspension to new culture vessels.
  6. Incubate culture vessels 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 (approximately 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.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended.
Medium Renewal: Every 2 to 3 days.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor R Nusse
References

Willert K, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448-452, 2003. PubMed: 12717451

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
References

Willert K, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448-452, 2003. PubMed: 12717451