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Following overnight growth, the biofilms were treated with nisin (10, 50 ��g/ml) with short exposure times (1, 5, 10 min). Following the treatment of pre-formed biofilms with or without nisin in CFS, all wells were washed with PBS three times. The same biofilm staining protocol was followed as described above. Confocal Laser Scanning Microscopy and Quantitative Analysis of Biofilms After nisin treatment, biofilms were imaged using Leica confocal laser scanning microscopy (CLSM, SPE, Leica, IL, USA) with a 40X1.25 NA HCX PL APO infinity-corrected oil or a HCX PL APO 40X/0.85 CORR CS objective. All biofilms were stained with the BacLight Live/Dead Bacterial Viability kit (Invitrogen, Carlsbad, MA, USA), which contains the nucleic acid stains Syto-9 (green signal) and propidium iodide (red signal) as described above. Once the microscopy images were taken, biofilms were rendered as 3D structures with Imaris (Bitplane, Zurich, Switzerland) computer imaging software. Image stacks were treated equally and the signal intensity of rendered 3D biofilm structures were confirmed using histograms generated in Imaris. Imaris allowed for the visualization of biofilm architecture in three dimensions, penetration of nisin into biofilms (inferred by the extent and degree of the red signal), and the preparation of 3D files for the quantification of biofilm structure using the computer software program Comstat2. For detailed biofilm analysis, Cisplatin Comstat2 was used to determine the biofilm biovolume (total amount of space/biomass occupied by a biofilm), average thickness (thickness of each biofilm extending from the bottom to the top of the growth/viewing channel surface), and roughness (a measure of heterogeneity in biofilm architecture). The degree of killing, based on green (Syto-9; live) and red (PI; dead/damaged) pixel intensity for every pixel in all 3D planes were evaluated using ImageJ (National Institutes of Health). The percentages of live to dead/damaged cells was determined by first multiplying the total number of pixels by the level of intensity (0�C255) and then summing the total value for both the LIVE and DEAD signals from each image stack recorded. All renderings and quantification analyses were performed on a dedicated laptop computer equipped with an Intel Core i5 CPU with 8 GB RAM, 64-bit operating system (MSI Computer Corp., Industry, CA, USA). Cell Culture A direct cell outgrowth technique was used to obtain primary periodontal ligament (PDL) cells and gingival fibroblast (GF) cells as previously described (Scanlon et al., 2011). These cells were maintained in minimum essential medium alpha (��-MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% fungizone (Gibco, Life Technologies, Grand Island, NY, USA). Passage 3 or 4 PDL and GF cells were used for experiments.