A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

Influence of mechanical and chemical degradation on surface gloss of resin composite materials

Purpose: To determine the changes in surface gloss of different composite materials after simulation of mechanical and chemical aging mechanisms. Methods: 36 specimens were fabricated for each material and polished with 120-, 220-, 500-, 1200-, 2400- and 4000- grit SiC abrasive paper, respectively. Gloss measurements were made with a glossmeter (Novo-Curve) prior to testing procedures. Specimens of each material were randomly divided into three groups. Group 1 was conditioned for 7 days at 37 degrees C in 75% ethanol aqueous solution. Group 2 was immersed in fluoride gel (Elmex Gelée) at 37 degrees C for 1 hour. Group 3 was subjected to simulated toothbrushing with an electric toothbrush while being immersed in toothpaste. Surface gloss measurements were made subsequently. Results: Significant difference between surface gloss of the composite materials tested were detected after simulated brushing (Kruskal Wallis, P < 0.05). With the exception of Filtek Silorane, all composite materials tested were significantly affected by immersion in Elmex Gelèe (Wilcoxon signed-rank test, P < 0.05). Immersion in 75% alcohol aqueous solution significantly affected surface gloss except natural enamel and Durafill (Wilcoxon signed-rank test, P< 0.05)

A long-term laboratory test on staining susceptibility of esthetic composite resin materials

Objective: To evaluate the color stability of composite resin types designed for esthetic anterior restorations when continuously exposed to various staining agents. Method and Materials: Thirty-six disk-shaped specimens were made of each of 12 composite materials (1 microfilled and 11 hybrid composites). After dry storage at 37°C for 24 hours in an incubator (INP-500, Memmert), the initial color of each specimen was assessed by a calibrated reflectance spectrophotometer (SpectroShade). Specimens were immersed in five staining solutions or dry stored (control). All specimens were kept in an incubator at 37°C for 99 days. Test solutions were changed every 14th day to avoid bacteria or yeast contamination. After 99 days of storage, spectrophotometric measurements were again performed and L*a*b* scores once more recorded to determine the color changes. Results: Wine proved to have the highest staining potential followed by coffee, tea, orange juice, and cola, which had the lowest staining potential. The highest color change measured against a white background was observed for Durafill (Heraeus Kulzer) in wine (ΔE = 62.3), while the least staining was found for Enamel HFO (Micerium) in cola (ΔE = 3.5). The highest color change measured against a black background was observed for EsthetX (Dentsply) in wine (ΔE = 46.0), while the least staining was observed for Enamel HFO in cola (ΔE = 2.5). Conclusion: Composite staining susceptibility proved to vary among composite structure and brands. Potential discoloration might be limited by dietary restriction based on such in vitro evaluation