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The cells retain many characteristics of cortical collecting duct (CCD) cells including morphology and CCD antigens.

Most cell lines cloned from M-1 exhibit characteristics of either intercalated cells (ICC) or principle cells (PC) of the CCD.

5 to 10% of the cells exhibit a dual PC - ICC phenotype.

When grown on permeable supports, the cells develop a lumen negative transepithelial potential difference.

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.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended
Medium Renewal: 2 to 3 times per week

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.

Fejes-Toth G, Naray-Fejes-Toth A. Differentiation of renal beta-intercalated cells to alpha-intercalated and principal cells in culture. Proc. Natl. Acad. Sci. USA 89: 5487-5491, 1992. PubMed: 1608958

Stoos BA, et al. Characterization of a mouse cortical collecting duct cell line. Kidney Int. 39: 1168-1175, 1991. PubMed: 1654478

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.