When bacterial count was more than 200, we determined it was positive

80% covered with gold microelectrodes that monitor the electronic impedance, detecting physiological changes of the cells. Cells in contact with the electrode will act as insulators, leading to an increase in impedance. Thus, the electrode impedance changes proportionally with alterations to number, size and adherence of cells MedChemExpress Luteolin 7-O-β-D-glucoside growing in a monolayer. Changes in impedance are translated as the unitless term cell index. CI = /15, where Zi is the impedance at an individual point of time during the experiment and Z0 is the impedance at the start of the experiment. Thus, the CI is a quantitative and composite measure of the overall state of the cells in an electrode-containing well. First, 100 mL of complete medium were added to each well for measurement of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689277 background. Then, LNCaP cells were seeded Distribution of F-actin LNCaP cells were grown on glass cover slips uncoated and coated with the indicated reagents for 24 h and 96 h. Cells were then fixed and permeabilized as described above, followed by staining with rhodamine-phalloidin and 1 mg/ mL DAPI. The immunofluorescence complexes were visualized with an Olympus confocal microscope using a 606 lens. Optical sectioning was carried out by acquiring a stack of images at different focal positions along the zaxis. Scratch wound assay LNCaP cells were seeded in a 96-well Essen ImageLock plate uncoated or coated as described previously, and were grown to confluence in a CO2 humidified incubator. After 24 h, the scratch was made using the 96-pin WoundMaker. Wound images were taken every 1 h for 36 h, and the data were analyzed by the Differential Effects of Coating Substrates on Prostate Cancer Cell integrated metric Relative Wound Density part of the live content cell imaging system IncuCyte HD. The experiment was done in triplicate. Sensitivity to simvastatin The influence of FN, PLO and PLL coating on the cell sensitivity to simvastatin was investigated. LNCaP cells were seeded in triplicate and grown in a 96-well E-plates as described above. After 24 h incubation, the cells were treated with different concentrations of simvastatin and monitored every 1 h for 60 h using the RTCA system. The IC50 for 24 h, 48 h and 72 h treatment were calculated using the software GraphPad Prism 5. qRT-PCR Surfaces of a 6 well-plate were coated as described above, and LNCaP cells were seeded at a density of 1.056104 cells/cm2. After 72 h, the growth media was substituted by charcoal-stripped serum RPMI media supplemented with 5% CSS and the cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692147 were cultured for 48 h. Finally, LNCaP cells were treated with 20% ethanol as control or the androgens R1881 and DHT for 30 h. Total RNA was obtained using the RNeasy mini kit according to the manufacturer’s instructions. The quantity and the quality of the RNA were measured using a NanoDrop UV spectrophotometer. Samples with a 260/280 ratio higher than 2.0 were used for subsequent procedures. The samples were treated with DNAse Amp grade I, and 2 mg of total RNA was reverse-transcribed using the cDNA synthesis method for the qPCR kit. QRT-PCR was performed with SYBR Green master mix using the 7900HT Fast Real-Time PCR System. Data were analyzed with SDS2.3 software. The mRNA expression levels were calculated by the DDCt method and normalized relative to the expression levels of the house keeping gene of the respective treatment and calculated relative to the ethanol uncoated control. The sequences of the primers used are listed in fibronectin resulted in the hi