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Cell Biology
ATCC® Number: CRL-2814™    Price: $365.00
Designations: L Wnt-5A
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
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 ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.
Comments: L-M(TK-) cells (ATCC CCL-1.3) were transfected with a Wnt-5A expression vector and stable clones were selected in medium containing G418. The Wnt-5A gene encodes a secreted glyoprotein with a variety of signaling effects. Wnt proteins bind to receptors of the Frizzled family on the cell surface. Wnt genes control many of the patterning and growth events during embryonic development. The cells secrete biologically active Wnt-5A protein. They are presently the best source for production of Wnt-5A conditioned medium. Since the conditioned medium contains other factors besides the Wnt-5A protein, it is necessary to control any experiments involving the Wnt-5A conditioned medium with control conditioned medium from the parental cell line (ATCC CRL-2648).
Propagation: ATCC complete growth medium: Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 0.6 mg/ml G-418 and 10% fetal bovine serum
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C
Subculturing: Protocol:
  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 gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    An inoculum of 5 X 10(3) to 3 X 10(4) viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C.

Protocol for Wnt-5A conditioned medium: 1. Split the cells 1:10 in 10 ml culture medium without G418 in 10 cm petri dishes 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 without G418 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-5A conditioned medium. It is stable at 4 degrees C and can be frozen.
Interval: Maintain cultures at a cell concentration between 7 X 10(3) and 3 X 10(5) cells/cm2
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended
Preservation: Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Doubling Time: 36 hrs
Related Products: Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2002
recommended serum:ATCC 30-2020
parental cell line:ATCC CRL-2648
References: 88964: Chen W, et al. Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4. Science 301: 1391-1339, 2003. PubMed: 12958364
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