To detect the percentages of GFP-positive cells, mock- or KSHV-infected MSCs were detached from the plate with 0.25% trypsin at 24 h postinfection and washed once with PBS. GFP-positive cells were quantified on a Guava easyCyte flow cytometer (Merck Millipore, Bedford, MA). Data were analyzed using InCyte 3.1 software (Merck Millipore).
To detect the expression of cell surface markers, cells detached from the plate with 5 mM EDTA in Dulbecco’s phosphate-buffered saline (PBS) were fixed with 4% paraformaldehyde in PBS for 20 min and incubated with primary antibodies at a 1:100 dilution for 30 min at 4°C. Mouse monoclonal antibodies to CD31 (Abcam), CD144 (eBioscience, San Diego, CA), vimentin (Abcam), and CD90 (eBioscience) and rabbit polyclonal antibodies to vWF (Dako, Carpinteria, CA), podoplanin (Abcam), and LIVE-1 (Abcam) were used. After being washed in PBS, cells were incubated with allophycocyanin (APC)-conjugated goat anti-mouse or -rabbit antibodies (R&D Systems, Minneapolis, MN). Flow cytometry was performed as described above.
To detect the expression of cell surface markers, cells detached from the plate with 5 mM EDTA in Dulbecco’s phosphate-buffered saline (PBS) were fixed with 4% paraformaldehyde in PBS for 20 min and incubated with primary antibodies at a 1:100 dilution for 30 min at 4°C. Mouse monoclonal antibodies to CD31 (Abcam), CD144 (eBioscience, San Diego, CA), vimentin (Abcam), and CD90 (eBioscience) and rabbit polyclonal antibodies to vWF (Dako, Carpinteria, CA), podoplanin (Abcam), and LIVE-1 (Abcam) were used. After being washed in PBS, cells were incubated with allophycocyanin (APC)-conjugated goat anti-mouse or -rabbit antibodies (R&D Systems, Minneapolis, MN). Flow cytometry was performed as described above.