The biofilm viability was then assessed by quantifying CFU

minutes. The acidic lysates were neutralized by initially adding 80 ml 2 M KOH/0.2 M KH2PO4, pH 7.5, and adjusting the pH by further addition of 15 ml buffer until reaching neutral pH. Extracts were centrifuged at 13,000 rpm at 4uC for 3 minutes. Supernatants were snap order Varlitinib frozen in liquid nitrogen and stored at 220uC until analysis. NAD+/ NADH was measured spectrophotometrically in an enzymatic cycling assay. The NAD+/NADH content of 10 ml sample was measured in 140 ml reaction mix containing 1.8 mM 3–2,5-diphenyl-tetrazolium bromide, 70 mM 1methoxy-5-methyl-phenzzinium methyl sulfate, 64 mM nicotinamide, 0.32 M ethanol, 64 mM Gl-Gly buffer, 20 mM succinate, and 20 U alcohol dehydrogenase. The NADP+/NADPH content of 10 ml sample was measured in 140 ml reaction mix containing 1.8 mM 3–2,5-diphenyl-tetrazolium bromide, 70 mM 1methoxy-5-methyl-phenzzinium methyl sulfate, 5 mM glucose-6phosphate, 50 mM Tris-HCl, 20 mM succinate, and 0.45 units glucose-6-phosphate dehydrogenase. After addition of reaction mixes, plates were incubated at 37uC for 30 60 minutes and absorbance measured at 510 nm on a BioRad Benchmark Plus micro plate reader. NAD and NADP concentrations were calculated from an NAD+ and NADP+ standard curve, respectively. Absorbance values were corrected for blank absorbance before concentrations were determined. All samples were corrected for the dilution factor. Glucose and Lactate Assays Glucose consumption measurements were based on the Amplex Red Glucose/Glucose Oxidase assay kit from Molecular probes. Glucose, glucose oxidase, and Amplex Red reagent were used from the kit but horseradish peroxidase was obtained from Sigma-Aldrich and 1x reaction buffer was replaced with 0.05 M Tris-HCl, pH 7.5. Apart from these minor changes, the kit protocol was followed as described by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19656604 the manufacturer. Lactate production was measured using the same protocol as for glucose consumption but replacing glucose oxidase with lactate oxidase and including a lactate standard series instead of glucose. Cells were seeded in 12 well tissue culture plates and incubated for 24 hours in either 1 ml control or 1 ml 5 nM FK866 medium. For glucose measurements, medium containing 5 mM glucose was used while lactate production was measured for cells grown in 25 mM glucose medium. After 18 hours, medium was refreshed in some wells for measurement of glucose consumption or lactate production during the last 6 hours of incubation. Prior to addition of incubation media, wells were always rinsed with pure DMEM containing no glucose. Medium was collected at the end of the 24 hour incubation period and supernatants were snap frozen in liquid nitrogen and stored at 220uC until analysis. Cytosolic extracts were prepared in lysis 100 buffer and total protein was determined with the Bradford assay. Glucose consumption was calculated by subtracting the amount of glucose in the sample from that in medium without cells. Lactate production was calculated by subtracting the concentration of any lactate in the medium without cells from that of the samples. Glucose and lactate assays were performed in parallel. ATP Assay Intracellular ATP was determined using the CellTiter-Glo cell viability assay kit from Promega. A total of 500,000 cells were seeded per well of a 6-well plate and treated with 5 nM FK866 for 1, 3, or 24 hours prior to assay. Cells were washed twice with ice cold PBS and then scraped in 350 ml ice cold 0.6 M perchloric acid. PCA extracts were centrifuged for