After thawing at room temperature, the stock was used as inoculum Buparlisib for monolayers of naïve C6/36 cells in Leibovitz’s (L-15) medium containing 1% heat-inactivated fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB) and 1.2% antibiotic (Penicillin G and Streptomycin). At day 4 after challenge, the supernatant solution was removed and used as inoculum for subsequent trials. Immunostaining for flow cytometry Cultured insect cells were fixed with 4% paraformaldehyde in phosphate-buffer saline (PBS) for 20

minutes at room temperature, washed twice with PBS and treated with 0.1% triton X-100 in PBS. They were incubated with monoclonal antibody against the capsid protein of AalDNV [1], 3H5 monoclonal antibody against DEN-2 envelope protein [6] and J93 monoclonal antibody against JE envelope protein. [antibodies were kindly provided by Ananda Nisalax at the USArmed Forces Research Institute of Medical Sciences (AFRIMS) Bangkok] at room temperature for 1 hour. They were washed again with 0.1% triton X-100 in PBS and incubated in a 50-fold

dilution of anti-mouse IgG rabbit immune serum conjugated with FITC (F0261, DAKO) for 30 min at room temperature in the dark. After incubation, cells were washed once, resuspended in 1% formaldehyde in PBS FDA-approved Drug Library concentration and analyzed using a FACScan flow cytometer (Becton Dickinson). Mock cells were run in parallel very and served as negative controls. At least 10,000 cells were gated by light scatter and collected in a list mode manner. Data analysis was performed using Cell Quest software

(Becton Dickinson). The percentage of positive cells was determined from FITC fluorescence histograms using a region defined according to mock cells. Immunofluorescent staining for confocal microscopy Cells from passage 16 were re-supended as described above and transferred for attachment to microscope slides. They were fixed with 4% paraformaldehyde in PBS for 15 min, washed twice with PBS, permeabilized with 0.1% Triton X-100 for 5 min and blocked with PBS containing 10% FBS. They were incubated for 1 hour with monoclonal antibody against the appropriate virus followed by incubation for 30 min with 1:500 dilution of fluorophore-labeled secondary antibody conjugate (Alexa Fluor 546 goat anti-mouse IgG, A-11001, from Molecular Probes) directed against the primary antibody. They were then washed with PBS before analysis. TO-PRO-3 iodide (T-3605, Molecular Probes) was used for nucleic acid counterstaining. Immunofluorescent-stained cells were analyzed by fluorescence microscopy and confocal laser microscopy (FV1000, Olympus). Two slides were prepared for each antibody assay. After scanning whole preparations to gain an overall impression, 6 representative fields were photographed (approximately 150 cells) in order to record the proportion of immunopositive cells. References 1.