General Maintenance and Reactivation of iSLK Cell Lines.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi's sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms. Key features • This protocol describes methods for culturing and antibiotically selecting iSLK-BAC16 cells for robust KSHV reactivation. • Use of flow cytometry to quantify KSHV reactivation rates. • Innovative use of automated plate readers to assess KSHV reactivation. Graphical overview Schematic overview of the procedures used for general iSLK-BAC16 cell culture and Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation of iSLK-BAC16 cells. A typical timeline of iSLK-BAC16 cell culture, antibiotic selection, KSHV reactivation, flow cytometry quantification, and plate reader assessment of KSHV lytic replication. Corresponding media requirements are denoted under the respective procedures. iSLK-BAC16 = doxycycline-inducible endothelial cells harboring the KSHV genome on an artificial bacterial chromosome; Dox = doxycycline; NaBu = sodium butyrate.