S. alphaherpesviruses. Herpesviruses (family for 2 h. After the supernatant was removed, infected cells were washed twice with phosphate-buffered saline, refed with the appropriate medium containing monensin, and incubated at 37C for a further 5 h. The cells then were fixed with 4% paraformaldehyde and analyzed by FACSCalibur with Cell Quest software (Becton Dickinson). Energy depletion. YK333 was bound to CHO-hPILR or CHO-hNectin-1 cells (at an MOI of 1 1) at 4C for 1 h. The inoculum was then removed, and the cells were treated with glucose-free medium containing 2% bovine serum albumin, 0.3% 2-deoxy-d-glucose, and 0.05% sodium azide or with control medium and held at 4C for 15 min and then at 37C for 30 min. Extracellular virus was acid inactivated by removing the medium and replacing it with acid citrate buffer (40 mM citric acid PBIT [pH 4.7], 135 mM NaCl, 10 mM KCl) for 2 min. The cells then were washed, refed with the appropriate medium, and incubated for a further 7 h. The cells were then fixed with 4% paraformaldehyde and analyzed PBIT by FACSCalibur. Electron microscopy (EM) analysis. CHO-hNectin-1 or CHO-hPILR cells or primary human CD14-positive PBMCs were mixed PBIT with HSV-1(F) (at an MOI of 50), followed by centrifugation at 4C at 1,100 for 2 h or 30 min, respectively, to allow attachment, and then incubated at 37C for 2 min. Infected cells were fixed with 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 1 h on ice. The cells then PBIT were harvested and fixed for 1 h with the same fixative. Small pieces of the fixed pellet were washed with the same phosphate buffer containing 3% sucrose and postfixed with 1% osmium tetroxide in the same phosphate buffer for 1 h on ice. For primary human CD14-positive PBMCs, infected cells were fixed with 2.5% glutaraldehyde in 0.1 M ice-cold sodium cacodylate buffer (pH 7.2) containing 0.5 mg ruthenium red/ml for 1 h, during which time cells were allowed to warm up to room temperature. The cells were then washed with 0.1 M cacodylate buffer (pH 7.2) and postfixed with 2% OsO4 in the same cacodylate buffer containing 0.5 mg ruthenium red/ml for 1 h at room temperature, followed by routine embedding in Epon. The samples were then dehydrated with an ethanol gradient series followed by propylene oxide, embedded in an Epon 812 resin mixture, and polymerized at 70C for 2 days. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with a Hitachi H7500 electron microscope. Virus entry assays. CHO cells or primary human CD14-positive PBMCs were inoculated with PRV or an HSV at an MOI of 5, followed by centrifugation at 32C at 1,100 for 2 h. The inoculum was then removed, and the cells were refed with the appropriate medium. (i) In experiments in which various types of CHO cells or primary human CD14-positive PBMCs were infected with recombinant HSVs carrying a fluorescent marker, infected cells were fixed with 4% paraformaldehyde at 8 h or 14 h postinfection, respectively, and analyzed by fluorescent microscopy or FACSCalibur. (ii) In experiments in which various types of CHO cells were infected with wild-type HSVs, infected cells were stained with anti-gD antibody at 8 h postinfection and fixed with 4% paraformaldehyde. The stained cells were then incubated with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) and analyzed by FACSCalibur. (iii) In experiments in which primary human CD14-positive PBMCs were infected with wild-type HSVs, infected cells were harvested at 14 h postinfection and analyzed Rabbit polyclonal to HOXA1 by immunoblotting (19) with anti-ICP27 antibody. (iv) In experiments in which various types of CHO cells were infected with PRV, infected cells were fixed with 4% paraformaldehyde at 8 h postinfection, permeabilized with 0.1% Triton X-100, and stained with anti-EP0 antibody. The stained cells were then incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen) and analyzed by PBIT FACSCalibur. (v) In experiments in which primary human CD14-positive PBMCs were infected with PRV, infected cells were harvested at 7 h postinfection and analyzed by immunoblotting with anti-EP0 antibody. Antibodies. Mouse monoclonal antibodies (MAb) to gD (DL6), ICP27 (8.F.137B), and -tubulin (DM1A) were purchased from Santa Cruz Biotechnology, Abcam, and.