rehydrated through an ethanol series to water, transferred to PBS, counterstained for 10 seconds in a 1:4 dilution of Mayer’s haemalum/water and mounted in 30% Glycerol in PBS. Primary Keratinocyte Culture Primary keratinocytes were isolated from back skin of adult mice. Hair from the dorsal skin of a mouse, killed by cervical dislocation, was removed with electric clippers and depilatory cream (Nair, Church & Dwight, New Jersey, US), applied to the skin surface for approximately 3 minutes. Excess cream was removed and the skin was washed under running water. Next, mice were washed twice for 2 minutes in Iodine solution, rinsed in sterile water and washed for 262 minutes in 70% ethanol. Skin was then cut from neck to tail and peeled off using forceps. Skin was placed, epidermis side down, on a sterile surface and the hypodermis was carefully scraped off using a scalpel blade. Next, the skin was stretched flat and a piece of autoclaved no. 1 filter paper (Whatman) was applied onto the exposed surface of the dermis. The skin, including the attached paper, was cut into 10 mm wide strips using sterile scissors and floated, dermis side down, on the surface of 1% Trypsin in PBS, and incubated for 2 h at 37uC. The epidermis was peeled off using curved forceps and placed into ice-cold PCT Epidermal Keratinocyte Medium containing 1.3 mM Ca2+ (Millipore, Watford, UK). The epider- mal sheets were dispersed in the medium using sterile scissors, until pieces were small enough to enter the tip of a 10 ml pipette. The resulting suspension was triturated by pipetting up and down 30 times, then transferred into a 50 ml conical tube on ice, leaving behind most of the cornified squames. The cell suspension was centrifuged at 2006g for 5 minutes. The cell pellet then was resuspended in high calcium medium (1.3 mM; CnT-07CF, Millipore) and filtered through a 100 mm cell strainer (BD Biosciences, Oxford, UK) into a new 50 ml conical tube. Cells were centrifuged at 2006g for 5 minutes and the cell pellet was resuspended in 10 ml high calcium medium. The cells were counted, centrifuged again and resuspended in the desired volume of low calcium medium (0.05 mM) to obtain a final cell concentration of 106 cells/ml. Cells were plated on Collagen I coated 60 mm dishes (VWR, Lutterworth, UK), at a concentration of 106 cells per dish. Primary keratinocytes were incubated in 7% CO2 overnight at 36uC. The next day, the medium containing unattached cells was aspirated, the dish was rinsed with calcium/magnesium free PBS (Sigma) and fresh low calcium media was added. Thereafter cells were maintained in low calcium medium, with medium changed every two days. In vitro Scratch Wound Assay Primary keratinocytes were seeded at a density of 106 cells per well in Collagen I pre-coated 6-well plates (VWR) and grown to near confluency, when the culture medium was replaced with starving medium (1% FCS) for 2 hours. Scratch wounds were made, using a sterile 200 ml micropipette tip to form a cross in each well, and medium was replaced with low calcium (0.05 mM) medium (CnT-07CF, Millipore) containing 8% calcium free FCS (Biosera, Ringmer, UK). To determine the rate of cell migration, images were acquired at 0, 12, 18 and 32 h post-wounding using a Time-lapse microscope set up (Zeiss). Scratch widths were measured at 20 points along their length using ImageJ software version 1.429 (National Institute of Health, Wayne Rasband, USA). The percentage of migration was determined relative to the scratch width at 0 hours. FGF22 in Skin Development and Repair In vivo Wound Healing Full-thickness excisional wounds were generated in shaved dorsal skin of 6–8 weeks old mice using a 3 mm biopsy punch. Wounds were examined daily and collected after 0, 1, 5, 14, and 21 days for analysis. Mice were housed in isolator cages of up to 3 mice, control and knockout animals were housed separately. For each time point, 3 female and 3 male mice of each genotype were analysed. One hour prior to culling, mice were injected with BrdU (0.25 mg/kg; Sigma). Wounds were excised together with 2 mm of surrounding tissue and one wound per mouse was placed on a nitrocellulose membrane (GE Healthcare) and fixed overnight in 1% Glacial Acetic Acid/99% Ethanol at 4uC for histological analysis. Morphometric analysis of wound repair was performed on Haematoxylin/Eosin stained paraffin sections using ImageJ software. The wound gap was calculated as the distance between two margins of the inward growing epithelium and was measured three times for each wound section. The area of epithelial tongue also was determined using ImageJ 1.429 software (National Institute of Health, Wayne Rasband, USA) to outline the entire wound epithelium, starting from the most wound proximal hair follicle. Skin Carcinogenesis Cohorts of seven weeks old female wild type and fgf22 knockout mice were subjected to two-step skin carcinogenesis protocol performed as described previously [19]. Female mice (n = 10 per group) were treated, at eight weeks of age, with topically administered DMBA (25 mg in 200 ml acetone; Sigma), or vehicle alone as a control. From 9 weeks old, all mice were treated weekly with TPA (7.4 mg in 200 ml acetone; Sigma) for 15 weeks. Mice were monitored on a daily basis and papilloma/tumour counts were recorded weekly for up to 58 weeks post-initiation, at which stage the experiment was terminated. If mice appeared sick or the lesion reached a predefined limit of 14 mm diameter, they were killed and examined post-mortem. Skin lesions and macroscopi- cally normal skin were harvested and fixed overnight in 10% Buffered Formalin for histological analysis. All animal experiments were approved by Cancer Research UK London Research Institute ethics committee and carried out under Home Office licence (70/5387, 70/5878), according to Institutional guidelines at Cancer Research UK London Research Institute. HaCaT Cells HaCaT cells, an immortalized human keratinocyte line (kind gift of Dr P. Boukamp; [27]), were cultured in DMEM supplemented with 10% foetal bovine serum at 37uC, and 8% CO2. Cells were passaged weekly in T75 flasks and all the experiments were performed with sub-confluent cells. HaCaT cells at 70% confluence were stimulated with hFGF7, hFGF10 (PeproTech, London, UK) or hFGF22 (Cambridge Bioscience) at a concentration of 100 ng/ml, co-treated with Heparin (300 ng/ml). FGF dependent stimulation was blocked by 30 minute pre-treatment with the FGFR specific inhibitor, PD173074 (1.7 mM; Sigma). Western Blot Analysis Cultured cells were lysed with NuPAGE LDS Sample buffer 2x (Invitrogen; supplemented with 100 mM DTT) for 5 minutes at room temperature, prior to scraping and brief sonication. Lysates were loaded in 4–12% gradient NuPAGE gels and run at 110 V at room temperature in NuPAGE MES running buffer (Invitrogen). Proteins were transferred onto nitrocellulose membranes (Schlei- cher & Schuell, Whatman) by electro-blotting for 3 h (4uC) at PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39436