Ion Currents at Cornea Wounds Figure 3. Calcium flux at corneal wounds. A. Ca2+ concentra- tion. Calcium concentration at unwounded cornea was slightly above background (0.015 mM). After wounding, Ca2+ concentraion increased until 20 min then plateaued. B. Ca2+ flux. Unwounded cornea showed a small Ca2+ efflux. After wounding, calcium efflux at the wound edge increased to reach a maximum value after 20 minutes. This efflux was maintained for up to 90 minutes. Aminophylline had no effect on Ca2+ flux. C. Fixation. Calcium concentration was measured for 30 minutes to confirm normal efflux. The eye was then fixed. Subsequent measurements showed a drop in calcium concentration to almost zero, even lower than unwounded values. doi:10.1371/journal.pone.0017411.g003 Cruz, CA, USA; Cat # sc-16430), was used to label the protein (1 hour at room temperature). After washing in PBS, the samples were incubated with Cy3 conjugated secondary antibody (1:200, Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and phalloidin-FITC (1:100, Sigma-Aldrich) for 1 h at room temperature. Cell nuclei were counterstained with 49,6-diamidino- 2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA; cat # H-1200). Flourescent signals were visualized on a fluorescent microscope (Axiovert 100; Zeiss, Germany). Quantitative PCR Expression level of CLC2 mRNA was determined using quantitative real time polymerase chain reaction (qPCR). PCR primers were designed using Primer3 online. Human CLC2 mRNA sequence was downloaded from the National Center for Biotechnology Information (NCBI) GenBank. A region that matched the consensus sequence for Figure 4. Potassium flux at corneal wounds. A. K+ concentra- tion. Immediately after wounding there was a rapid transient increase of K+ concentration. This initial high concentration decreased over 5–20 min after wounding, reaching a stable lower value. B. K+ flux. There was a large K+ efflux immediately after wounding, which dropped after 20 min. Aminophylline had no effect on the initial peak of efflux, but appeared to enhance efflux at later time points (60–90 min; *P,0.03). C. K+ concentration in high [K+]. In hi-K+ solution (20 mM) the initial peak of K+ concentration was absent and the wound showed a lower K+ concentration (negative values), indicating K+ influx. doi:10.1371/journal.pone.0017411.g004 CLC2 was chosen for PCR primers. Primers were chosen to be 20 bp in length, with a GC content of 45–60%, no long repeats of a single base, and 150–500 bp of products length. We further confirmed lack of cross-reactivity between the 39 end of the CLC- primers and other known genes by BLAST (basic local alignment search tool) searches. The optimized primer oligos used were synthesized by Eurofins MWG Operon (London, UK). Primary antibody against CLC2 was obtained from Santa Cruz Biotechnology, Inc. Human corneal epithelial cells were cultured in supplemented hormonal epithelial medium (DMEM/ F12; Invitrogen, Carlsbad, CA, USA) in 10 cm tissue culture dishes at 37uC and 5% CO2 until confluent. Scratch wounds were made with a sterile plastic pipette tip. Vibrating probe We have previously described in detail the vibrating probe technique for non-invasively measuring endogenous electric current [7,37]. Briefly, the probe is an insulated stainless-steel PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17411