HaCaT keratinocytes were grown to 90% confluence on 18 mm2 glass cover slips placed in six-well plates. Keratinocytes were then exposed to 2 ml BCM, PCM, or EPI. At 4 or 24 hours, apoptotic ACY-1215 concentration keratinocytes were
detected using the APO-BrdU TUNEL Assay Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s staining protocol as previously described. Cells were counter stained with propidium iodide. Coverslips were imaged using a Nikon Eclipse E800 epifluorescent microscope using a 10 × objective. For analysis, four images of each condition were taken and numbers of adherent cells staining positive for TUNEL and propidium iodide were counted and the percentage of cells staining positive for TUNEL were calculated. Acknowledgements This work was supported by grant number 1P20GM078445-01 from SAHA HDAC nmr the National Institute of General Medical Sciences (NIGMS). The contents of this project are solely the responsibility of the authors and do not necessarily represent
the official views of the NIGMS. We would like to thank Laura Jennings and Al Parker for helpful discussions on manuscript preparation and statistical analysis, respectively. Electronic supplementary material Selleck Temsirolimus Additional file 1: Genes significantly regulated in BCM treated HKs. Transcriptional profile (fold change ±1.5, pval < 0.01 BCM relative to PCM) of HKs after four Vasopressin Receptor hours of exposure. (PDF 79 KB) References 1. Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG: Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 2002,184(4):1140–1154.PubMedCrossRef 2. Resch A, Rosenstein R, Nerz C, Gotz F: Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. Appl Environ Microbiol 2005,71(5):2663–2676.PubMedCrossRef 3. Stewart PS, Costerton JW: Antibiotic resistance of bacteria in biofilms. Lancet 2001,358(9276):135–138.PubMedCrossRef 4. Zhu J, Miller MB, Vance RE, Dziejman M, Bassler BL, Mekalanos JJ: Quorum-sensing
regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 2002,99(5):3129–3134.PubMedCrossRef 5. Cotter PA, Stibitz S: c-di-GMP-mediated regulation of virulence and biofilm formation. Curr Opin Microbiol 2007,10(1):17–23.PubMedCrossRef 6. Fux CA, Costerton JW, Stewart PS, Stoodley P: Survival strategies of infectious biofilms. Trends Microbiol 2005,13(1):34–40.PubMedCrossRef 7. Wolcott RD, Kennedy JP, Dowd SE: Regular debridement is the main tool for maintaining a healthy wound bed in most chronic wounds. J Wound Care 2009,18(2):54–56.PubMed 8. James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, Costerton JW, Stewart PS: Biofilms in chronic wounds. Wound Repair Regen 2008,16(1):37–44.PubMedCrossRef 9.