In case of lacking or reduced tightness of the monolayer, very high counts were

Unless otherwise noted, mycobacterial strains were cultured in 7H9 media containing 0. 5% glycerol, 0. 05% tyloxapol, and 10% OADC. Cell culture, infection, and mycobacterial survival Murine RAW264. 7 macrophages have been described MAPK 癌 pre viously. Cells were cultured in DMEM supplemented with 10% heat inactivated fetal bovine serum, L glutamine, sodium pyruvate, and non essential amino acids. Human A549 alveolar epithelial cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% sodium pyruvate, 1% HEPES and 1% of both non essential and es sential amino acids. For infections, mycobacteria growing in 7H9 OADC were washed in PBS with 0. 05% tyloxapol, sonicated to minimize bacterial clumping, and adjusted to the multiplicity of infection 5. RAW264.<br><br> 7 cells were first infected with mycobacteria, chemical inhibitors were added for the indicated periods of time, and CFU MK-1775 955365-80-7 was then determined. For assays involving bone mar row derived macrophages, the bone marrow was isolated and depleted of red blood cells. The cells were then differentiated toward the macrophage lineage with DMEM media supplemented with L929 derived culture supernatant. Infection and CFU determination were performed as described above. Western blots The following antibodies were used in this study, anti Actin plus anti mouse HRP were purchased from Gen Script and Anti phospho S6, anti S6, and anti rabbit HRP were purchased from Cell Signaling. Blotting conditions and chemiluminescence have been described. DQ BSA assays and flow cytometry RAW264. 7 macrophages were loaded with DQ BSA as described previously.<br><br> After an overnight treatment with mTOR inhibitors, DQ BSA was detected utilizing the PE channels of a BD FACSCanto or a BD LSRII flow cytometer. Flow cytometry was performed at both the Duke University Shared Cytometry Resource and the Yale School of Medicine buy MS-275 Cell Sorter Facility. The data were analyzed with FlowJo software. LC3 Immunofluorescent staining A549 cells were cultured on glass coverslips, fixed with 4% formalin, stained with either rabbit anti LC3B or isotype control, and visualized with Alexa Fluor 594 conjugated anti rabbit secondary antibodies as described. Counter staining with Hoechst, fluores cent microscopy, and image analysis were performed as described previously. Statistics Analysis was performed with GraphPad Prism using an analysis of variance with a Tukey post test or Students T test.<br><br> P values of p 0. 05 were considered to be significant. In recent years, the kinase field has developed the prac tice of monitoring inhibitor selectivity through profiling on panels of biochemical assays, and other fields are following this example. Such profiling means that scientists are faced with increasing amounts of data that need to be distilled into human sense. It would be powerful to have a good single selectivity value for quantitatively steering the drug discovery process, for measuring progress of series within a program, for com putational drug design, and for establishing when a compound is sufficiently selective. However, in contrast to, for instance, lipophilicity and potency, where values such as logP or binding constant are guiding, quantitative measures for selectivity are still under debate.