Isolation of mouse epidermal keratinocytes and their in vitro clonogenic culture

Rebecca J. Morris, Nyssa Readio, Kelsey Boland, Kelly Johnson, Sonali Lad, Anupama Singh, Ashok Singh, Stephanie Holtorf, Samantha Skaar

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 ± 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

Original languageEnglish (US)
Article numbere58701
JournalJournal of Visualized Experiments
Issue number150
StatePublished - Aug 2019

Bibliographical note

Publisher Copyright:
© 2019 Journal of Visualized Experiments.


  • 3T3 fibroblast
  • Adult mouse epidermis
  • Biology
  • Cell culture
  • Clonal culture
  • Epidermal keratinocytes
  • Issue 150
  • Keratinocyte stem cells


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