Live cell imaging: Fluorescent dyes, such as SYTOX green (Molecular Probes), which is a high-affinity nucleic acid stain but cell-impermeable dye (Ex./Em = 500nm/523nm), will be used to examine cell viability during or after treatment. Such dyes will enter cells with compromised plasma membranes but will not those of live cells. Using a 40x or 60x water-immersion objective lens immediately after mixing the viruses and host cells, dual-excitation and dual-emission images will be collected simultaneously under a BioRad MRC-1024ES confocal laser scanning microscope equipped with Argon/Krypton lasers. Series of such images will be collected using a computerized time-course program (BioRad LaserSharp program). Quantitative analyses of images from at least three independent experiments of duplicate cell cultures will be performed to determine the effect of ???? on the rate of cell survival or apoptosis.
Immunochemical labeling: Cells growing in 8-well chambers exposed to different treatments will be fixed in 4% paraformaldehyde and will be used for immunochemical assays for apoptosis-related agents or proteins using standard single or double labeling immunofluorescence microscopy.