L Cells (ATCC® CRL-2648)

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

Organism Mus musculus, mouse
Tissue subcutaneous connective tissue; areolar and adipose
Cell Type fibroblast
Product Format frozen
Morphology fibroblast
Culture Properties adherent
Biosafety Level 1
Age 100 days
Gender male
Strain C3H/An
Applications
This cell line is a suitable transfection host.
It is used to obtain control conditioned medium for comparison to Wnt-3A or Wnt-5A conditioned medium from L Wnt-3A cells or L Wnt-5A cells.
Storage Conditions liquid nitrogen vapor phase
Clinical Data
male
100 days
Comments
It is the parental line for the L Wnt-3A cell line (ATCC CRL-2647) and the L Wnt-5A cell line (ATCC CRL-2814).
The cell line named L Cells is closely related or identical to L-M(TK-) cells (ATCC CCL-1.3).
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: 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 Control Conditioned Medium:

  1. Split the cells 1:10 in 10 mL culture medium 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 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 L Cell 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

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