Isolation of Epidermal Progenitors from Mouse Skin

Methods for preparing embryonic mouse back and head skin for fluorescence activated cell sorting (FACS) and purification of α6-high epidermal and hair bud progenitors have been previously described (Williams et al., 2011 (link)). Briefly, the skin of E14.5 and E17.5 embryos was dissected and either (E14.5) placed directly into a trypsin-EDTA solution at 37°C for 5 min on an orbital shaker, or (E17.5) first treated with the enzyme dispase (Gibco, 1:1 in PBS) overnight at 4°C prior to making the single cell suspension. Sorting buffer (PBS 5% FBS) was added to the suspension to neutralize trypsin. Single-cell suspensions were obtained by filtering through a 70 µM strainer and collected by centrifugation at 300 g for 5 min. Cell suspensions were washed three times and incubated with the appropriate antibodies for 30 min on ice. For FACS, we used the following antibodies (along with epifluorescent markers): α6-integrin (eBiosciences) to select for basal progenitors, CD140a (PDGFRA) (eBiosciences) to select against mesenchymal cells, CD31 (PECAM1) (eBiosciences) to select against platelets. DAPI was used to exclude dead cells. Cell isolations were performed on FACS Aria sorters running FACS Diva software (BD Biosciences).