Expression profile of Gli1-expressing keratinocytes from mouse skin

To assess if Hedgehog (Hh) responding cells in the skin have a unique expression profile, isolated keratinocytes that express the Hh response gene Gli1 were collected by FACS and their gene expression was compared to sorted CD34-expressing cells from the middle bulge region of the hair follicle and to cells from the interfollicular epidermis (IFE) by hybridization of isolated RNA to gene expression microarrays. Dissociated keratinocytes isolated form back skin of Gli1-eGFP/+ mice (2 mice pooled for each replicate) in adult telogen were sorted based on GFP expression and immunostaining for CD34. Only viable, single cells with immunostaining for α6 Integrin (to mark basal keratinocytes) were collected. GFP(+) cells were collected as the Gli1 cohort. GFP(-) CD34(+) cells were collected as the CD34 cohort, and GFP(-) CD34(-) cells were collected as interfollicular epidermis (IFE) cohort. Total RNA was extracted from each cell population and labeled for hybridization to gene expression microarrays. The experiment was preformed in triplicate, however RNA from the Gli1(+) cells in one replicate was of insufficient quality to analyze. For staining of isolated keratinocytes, cells were incubated on ice for 1 hour in SMEM containing 1%BSA and 15μL of anti-CD49f-PE antibody (BDBioscience) plus 5μL anti-CD34-APC antibody (eBioscience) for each 100μL total volume for each 2 million cells. Immediately prior to sorting, DAPI (Invitrogen) was added to the washed cells at 1μg/mL final concentration.