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[28] H1N1pdm09 (H1N1A/Auckland/3/2009) was obtained from the World Health Organization (Melbourne). Viral stocks were propagated in Madin�CDarby canine kidney cells (ATCC, Manassas, VA, USA), and stock viral concentrations were measured using plaque assays, as described previously.[25] Whole blood was collected in serum tubes and sent to Hunter Area Pathology Service (Newcastle, NSW, Australia) for standard neutrophil counts. Whole blood was collected in ethylenediaminetetraacetic acid tubes, and PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Uppsala, Sweden), as per the manufacturer's instructions. PBMC were resuspended in Roswell Park Memorial Institute media (Invitrogen, Australia) with 5% foetal bovine serum (SAFC Biosciences, Lenexa, KS, USA) and plated in 24 well plates (Thermo Scientific Nunc Scoresby, Victoria, Australia) at a final concentration of 2.0?��?106?cells/mL. PHA (Sigma-Aldrich, Tenofovir solubility dmso St. Louis, MO, USA) was used at a final concentration of 5?��g/mL, and H1N1pdm09 stock was added a final concentration of 0.1?pfu/mL. PBMC were cultured for 48?h at 37��C in 5% CO2, with no media changes during this period. Cultures were centrifuged at 550?��?g for 10?min and supernatant was stored at ?80��C for protein analyses. XAV 939 Cell viability was assessed by PE Annexin V Apoptosis Kit I (BD Bioscience, San Jose, CA, USA), as per the manufacturer's instructions. IFN-��, IL-10 and IL-17 protein were measured from culture supernatant as per the manufacturer's instructions (BD Bioscience). Samples were run on a BD FACS Canto II Flow Cytometer and analysed with BD FCAP Array Software (BD Bioscience). The minimal limits of detection were 1.8?pg/mL (IFN-��), 0.13?pg/mL (IL-10) and 0.3?pg/mL (IL-17). The statistical packages STATA 11 (Stata Corp LP, College Station, TX, USA) and Graph Pad Prism4 (La Jolla, San Diego, CA, USA) were used. Normality of the data was tested using the Kolmogorov�CSmirnov test (with Dallal�CWilkinson�CLillie for P-value). To determine significant differences between group characteristics, the one-way analysis of the variance (or Kruskal�CWallis test) with multiple comparisons was used for continuous data and Fisher's exact test for categorical data. tuclazepam Group comparisons for the protein data were analysed using the Kruskal�CWallis test, followed by Dunn's multiple comparison test. If the levels of IL-17 were below detection, they were arbitrarily assigned the value of 0.1?pg/mL similar to the method of analysis previously described by Brooks et?al.[10] Correlations were made using Kendall's tau. Significance was accepted when P?