After thawing at room temperature, the stock was used as inoculum selleck 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 CYT387 in vivo and analyzed using a INCB28060 in vitro FACScan flow cytometer (Becton Dickinson). Mock cells were run in parallel pheromone 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.