Keratinocytes were isolated as previously described from punch biopsies of the skin from the buttocks region of AD DC and normal donors (26 (link), 32 (link)). The punch biopsies were from third and a second generation adult DC subjects (DC-HSK-1 and DC-HSK-4). The studies were approved by the University of Iowa Institutional Review Board for human studies and conformed to protocols outlined by the Declaration of Helsinki. Donors gave their written, informed consent for collection of tissue. Normal sex and age (within 1 year) matched biopsies were used as controls (referred to as N-HSK-1 and N-HSK-2). Briefly, the tissue was incubated overnight in dispase and on the following day, the epidermal layer was treated with trypsin to dissociate epithelial cells. The cells were plated on irradiated J2 3T3 feeders in E media as described (33 (link), 34 (link)). When 80% confluent, the cells were passed 1:4 (approximately 2 population doublings per passage). Passaging of cells was done by differentially removing feeder fibroblasts with a low percent trypsin/EDTA followed by complete trypsinization of remaining epithelial cells. For certain procedures, such as isolation of DNA, epithelial cells were passaged into keratinocyte serum free media (KSFM) for two passages to remove feeders before collection of cells.