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Clone C (ATCC® CRL-2531)

Organism: Oryctolagus cuniculus, rabbit  /  Cell Type: intercalated; SV40 large T antigen transfecte  /  Tissue: kidney, cortex  / 

Permits and Restrictions

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Organism Oryctolagus cuniculus, rabbit
Tissue kidney, cortex
Cell Type intercalated; SV40 large T antigen transfecte
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2 Cells contain SV40 viral sequences

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Applications
Cells were transfected by electroporation with the pZipSVtsA58 plasmid encoding a temperature-sensitive large T antigen of SV40 plus the neomycin resistance gene under the control of an SV40 promoter.
They express appropriate ultrastructural features, bind peanut lectin in an apical pattern, are rich in carbonic anhydrase and stain for proton-adenosinetriphosphatase in a basolateral pattern.
The cell line may be used to study terminal differentiation of intercalated cells.
Storage Conditions liquid nitrogen vapor phase
Derivation
Primary cultures of rabbit, bicarbonate-secreting, intercalated cells were isolated.
Following transfection, a line of G418 resistant cells was obtained and designated IC250. These cells were subcloned (Clone C) and characterized.
Comments
Primary cultures of rabbit, bicarbonate-secreting, intercalated cells were isolated.
Cells were transfected by electroporation with the pZipSVtsA58 plasmid encoding a temperature-sensitive large T antigen of SV40 plus the neomycin resistance gene under the control of an SV40 promoter.
Following transfection, a line of G418 resistant cells was obtained and designated IC250. These cells were subcloned (Clone C) and characterized.
The cells divide continuously at the permissive temperature of 32C. At the restrictive temperature of 40C, they cease dividing and assume morphological and transport properties of true bicarbonate-secreting intercalated epithelia cells.
They express appropriate ultrastructural features, bind peanut lectin in an apical pattern, are rich in carbonic anhydrase and stain for proton-adenosinetriphosphatase in a basolateral pattern.
These cells reverse the polarity of several proteins and show different cytoskeltal organization depending on the seeding density.
The cell line may be used to study terminal differentiation of intercalated cells.
Complete Growth Medium The base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006.To make the complete growth medium, add the following components to the base medium:
  • 0.005 mg/ml insulin
  • 0.005 mg/ml transferrin
  • 1.75 ng/ml selenious acid
  • 15 ng/ml epidermal growth factor(do not filter)
  • 25 ng/ml hydrocortisone
  • 10% fetal bovine serum

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-0.53mM EDTA solution to remove all traces of serum which 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 32°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.
  6. Incubate cultures at 32°C.

Subcultivation Ratio: 1:4 to 1:6
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

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Culture Conditions
Temperature: 32°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
Name of Depositor Q Al-Awqati
Deposited As rabbit
References

van Adelsberg J, et al. Isolation and culture of HCO3- -secreting intercalated cells. Am. J. Physiol. 256: C1004-C1011, 1989. PubMed: 2541617

Edwards JC, et al. Conditional immortalization of bicarbonate-secreting intercalated cells from rabbit. Am. J. Physiol. 263: C521-C529, 1992. PubMed: 1325122

van Adelsberg J, et al. An induced extracellular matrix protein reverses the polarity of band 3 in intercalated epithelial cells. Cell 76: 1053-1061, 1994. PubMed: 8137422

Vijayakumar S, et al. Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation. J. Cell Biol. 144: 1057-1067, 1999. PubMed: 10085301

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

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

van Adelsberg J, et al. Isolation and culture of HCO3- -secreting intercalated cells. Am. J. Physiol. 256: C1004-C1011, 1989. PubMed: 2541617

Edwards JC, et al. Conditional immortalization of bicarbonate-secreting intercalated cells from rabbit. Am. J. Physiol. 263: C521-C529, 1992. PubMed: 1325122

van Adelsberg J, et al. An induced extracellular matrix protein reverses the polarity of band 3 in intercalated epithelial cells. Cell 76: 1053-1061, 1994. PubMed: 8137422

Vijayakumar S, et al. Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation. J. Cell Biol. 144: 1057-1067, 1999. PubMed: 10085301

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.