Expression data from human keratinocytes after scratch wounds in vitro

In skin homeostasis, dermal fibroblasts are responsible for coordinating the migration and differentiation of overlying epithelial keratinocytes. As hairy skin heals faster than non-hairy skin, we hypothesised that follicular fibroblasts would accelerate skin re-epithelialisation after injury faster than interfollicular fibroblasts. We found that hair follicle dermal papilla fibroblast conditioned media (DPFi CM) could significantly accelerate wound closure compared to controls partly due to the presence of sAXL in this media. We used microarrays to identify upregulated and downregulated genes in human epidermal keratinocytes incubated with sAXL, DPFi CM and Epilife (keratinocyte growth media;control) after scratch wounds in vitro. 6 well plates were prepared by coating them using the coating matrix kit. Keratinocytes from two patients were seeded at a density of 6000 cells/cm2 using Epilife supplemented with EDGS. At confluency, a p200 pipette tip was used to scratch the well in 4 different regions (in a hashtag), to create a ‘wound’ in the cells. The cells were then washed two times with PBS to remove debris. Conditioned media obtained from DPFi, sAXL, and control with just Epilife supplemented with EDGS were added on the wounded cells. After 6 hours, media was removed, cells were washed in PBS, then RNA was collected using the RNeasy Plus Micro Kit (Qiagen). RNA was used to synthesize first-strand complementary DNA (cDNA) using Nugen Ovation V2. This was then converted to double-stranded cDNA, and used as a template for in vitro transcription to generate cRNA using the Nugen Encore Biotin Module. The cRNA was then transferred for hybridization and scanning onto the GeneChip™ Human Genome U133 Plus 2.0 Array.