Primary Mouse Keratinocyte Isolation

The Vero (ATCC) and HaCaT (obtained from Dr. Meenhard Herlyn, Wistar Institute, Philadelphia, PA) cell lines were maintained using standard procedures in DMEM supplemented with 10% fetal bovine serum and Gentamicin. Primary mouse keratinocytes were isolated from 24 to 72 h old pups^{20 (link)}. The pup tail tips were boiled in 10 mM NaOH, 0.1 mM EDTA, neutralized with 1 M Tris, pH 8.0 and used for PCR typing of sex with primers SMCX-1, 5′-CCGCTGCCAAATTCTTTGG-3′ and SMC4-1 5′-TGAAGCTTTTGGCTTTGAG-3′. These primers generate 1 product from female cells and 2 products from male cells. Skins were floated in 0.25% Trypsin (Invitrogen) overnight at 4 °C. The epidermis and dermis were separated and keratinocytes from the epidermis dissociated by mechanical shearing. Keratinocytes were maintained in a 1:1 mix of Keratinocyte-SFM with calcium and Keratinocyte-SFM without calcium (Invitrogen) supplemented with 10 ng mL⁻¹ mouse epidermal growth factor (Sigma-Aldrich), 200 μg mL⁻¹ bovine pituitary extract (Sigma-Aldrich) and Gentamicin. Experiments were performed with independent duplicate samples per timepoint and treatment. These duplicate samples were analyzed separately.