As a control was added to the same quantity of 136 and plaque assayed. The data shown is representative of 3 independent experiments. Each data point is the average of 3 replicates �� the standard deviation. A549 cells were detached and counted, and GDC-0941 customer reviews incubated with X-31 for 1 hour at 4. After washing with cold medium, the cells were fixed with 4 formaldehyde at RT. After washing, the cells were incubated in PBS containing 0.1 saponin, 1 bovine serum albumin, and HA mouse monoclonal antibody HA1 for 1 hour at room temperature. Cells were incubated with secondary anti mouse IgG-AF647 for 30 minutes. 10,000 cells were analyzed using FACS Canto II . Similar results were obtained when binding was performed on non-detached A549 cells. X-31 influenza virus was preincubated for 30 minutes with 2.5 ��M136 or 211. For the acidification assay, A549 cells were bound with X-31 for 1 hour at 4 in duplicate. Cells were warmed to 37 to allow endocytosis of the virus. After 1 hour the cells were washed with phosphate buffered saline , harvested by trypsinization, and fixed with 4 formaldehyde. After washing, the cells were incubated in PBS containing 0.1 saponin, 1 bovine serum albumin, and HA post-acid conformation α-Amanitin specific mouse monoclonal antibody A1 for 1 hour at room temperature. Cells were incubated with secondary anti mouse IgG-AF488 for 30 minutes. 10,000 cells were analyzed using FACS Canto II . Bafilomycin A1 was used at 50 nM as a negative control. The experiment was repeated three times. For the lipid mixing assay, X-31 virus was labeled with R18/SP-DiOC18 . A549 cells were infected for 1 hour as above. For the acid-bypass assay, trypsinized A549 cells were bound with labeled X-31 virus for 45 min on ice, and subsequently incubated for 2 min at 37 in either pH 6.8 or pH 5.0 medium, and fixed. For FACS analysis the cells were harvested by trypsinization and 5,000 cells were analyzed. For microscopy analysis, cells were grown on coverslips, infected, fixed in 4 formaldehyde, and examined using a Zeiss LSM 510 confocal fluorescence microscope. BafA was used as a negative control. BafA blocks endosome acidifi
