PHFAs were cultured on poly-L-lysine-coated 2-chamber glass slides, and the infection was performed as described above. The procedure of immunofluorescence assay has previously been described. Briefly, PHFAs infected with or without HHV-6A were fixed in 4% paraformaldehyde (in PBS), permeabilized in 0.5% Triton X-100 (in PBS), and stained with the anti-gp60/110 monoclonal antibodies (Chemicon international) followed by secondary antibody labeled with fluorescein isothiocyanate (FITC).
Electron microscopy
Cells were fixed with 2.5% glutaradehyde at room temperature for 1 h. After washing with PBS, the cells were collected, dehydrated in a series of 70%, 80% and 90% ethanol, and embedded in Epon. Ultrathin sections were cut and mounted in nickel grids, stained with uranyl acetate and lead citrate, and examined by a transmission electron microscopy.
Annexin V-propidium iodide (PI) staining
Apoptosis was measured by Annexin V-propidium iodide (PI) staining and flow cytometry. Infected and uninfected PHFAs were trypsinized, washed in PBS and incubated with Annexin V-FITC and PI solution (Bender MedSystems, Burlingame) in the dark for 15 min. Samples were analyzed by flow cytometry with FACSCalibur and BD CellQuest Pro software (Becton Dickinson, Mountain View). The amount of early apoptosis and late apoptosis was determined as the percentage of Annexin V+/PI- and Annexin V+/PI+, respectively.
Analysis of activated caspase-3 by flow cytometry
Caspase-3 activities in HHV-6A-infected and mock-infected PFHA were tested by flow cytometry with FITC-DEVD-FMK that recognizes cleaved caspase-3 according to the protocol by the manufacturer (Biovision Inc.).
Analysis of caspase-8 and caspase-9 using a colorimetric method
The activation of caspase-8 and caspase-9 was analyzed using a colorimetric assay kit (KeyGEN). Briefly, mock-infected and HHV-6A-infected PFHA were collected and resuspended in 50 μl of lysis buffer and incubated on ice for 30 min. After centrifugation, the protein concentration was assayed by the BCA Protein Assay kit (Byotime), and 50 μg protein was diluted in 50 μl lysis buffer for each assay. Then 5 μl of casepase-8 or caspase-9 substrate were added, respectively. The reaction mixture was incubated at 37°C for 4 h. The released chromphore was measured at 405 nm by a microplate reader.
Preparation of cytosolic and mitochondrial extracts
Cells were washed twice with PBS and kept for 1 h in ice-cold hypotonic buffer (20 mM HEPES, pH7.4, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, aprotinin and pepstatin) containing 250 mM sucrose. The cells were homogenized using a Dounce homogenizer (KONTES), and cytosolic and mitochondrial extracts were isolated as described previously.