Screenfect a plus

The iSLK cells (1 × 10⁵ cells) were transfected with either pCI-neo-3xFLAG empty plasmid or 3xFLAG-tagged ORF7 plasmid using Screenfect A plus (Wako, Tokyo, JAPAN), according to the manufacturer’s instructions, then simultaneously cultured with medium containing 8 μg/mL of Dox and 1.5 mM of NaB to induce lytic replication. After 3 days, culture supernatant was harvested and KSHV genomes were evaluated using real-time PCR. In addition to measuring KSHV genome, the virus titers in culture supernatants were also evaluated. The iSLK cells (1 × 10⁶ cells) were transfected with ORF7-3xFLAG plasmid using Screenfect A plus (Wako) and treated with Dox and NaB. After 72 h, culture supernatants were collected and centrifuged at 15,000 rpm 10 min at room temperature, then each supernatant (200 μL) was mixed with 1 × 10⁴ trypsinized HEK293T or HeLa cells. Polybrene (Sigma-Aldrich, MO, USA) was added to the cell mixture (8 μg/mL final concentration), and cells were placed into a 96-well plate. The 96-well plate was centrifuged at 1200× g for 1 h at room temperature. After 24 h, GFP-positive cells were counted by fluorescence microscopy.

Cells were transfected with the respective plasmids using a Screen Fect A or Screen Fect A Plus transfection kit (Fujifilm, Tokyo, Japan), in accordance with the manufacturer’s instructions. The medium was replaced 4 h after transfection. Transfected cells were generally used for experiments 48 h after transfection. Otherwise, G418 (2500 microgram/mL)-resistant clones were collected as stable clones.
We confirmed that FBD-102b cells were viable under each transfection experimental condition by verifying that attached trypan-blue-incorporating cells made up less than 5% of all cells in each culture.

Cells were transfected with the respective plasmids using a ScreenFect A or ScreenFect A Plus transfection kit (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. The medium was replaced 4 h after transfection. Transfected cells were generally used for experiments 48 h after transfection in biochemical experiments. We confirmed that FBD−102b cells were viable under each experimental condition by verifying that the attached trypan-blue (Nacalai Tesque)-incorporating cells made up less than 5% of all the cells in each culture.