Isolation and short term culture of primary keratinocytes, hair follicle populations, and dermal cells from newborn mice and keratinocytes from adult mice, for in vitro analysis and for grafting to immunodeficient mice.

Abstract

Protocols for preparing and culturing primary keratinocytes from newborn and adult mouse epidermis have evolved over the last 35 years. The present protocol is now routinely applied to mice of various genetic backgrounds for in vitro studies of signaling pathways in differentiation and in cell transformation, and for assessing the in vivo phenotype of altered keratinocytes in grafts of cells on immunodeficient mice. Crucial in the development and application of the procedure was the observation that keratinocytes proliferate in media of low calcium concentration, but rapidly commit to differentiation at calcium concentrations greater than 0.07 mM after the initial attachment period. Preparing primary keratinocytes from 10 newborn mice requires from 2 to 3 hours of hands-on time. Related procedures are also provided: preparing immature hair follicle buds, developing dermal hair follicles and fibroblasts from newborn mice, preparing primary keratinocytes from adult mice, and grafting cell mixtures on athymic nude mice.