ID8 is a cell line derived from mouse ovarian surface epithelial cells that have the ability to form extensive peritoneal tumors in vivo and have no known genetic mutations [24 (link)]. ID8 cells have variable ability to perform oxidative phosphorylation and glycolysis (glutamine independent) [25 (link)]. The ID8 cells used in this study were a gift from Dr. Vince Tuohy.
ID8 (syngeneic) EOC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% heat-inactivated FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1), which were grown under standard conditions. For luciferase transduction, in short, HEK293T/17 (ATCC CRL-11268) cells were plated and co-transfected with Lipofectamine 3000 (L3000015 Invitrogen, Waltham, MA, USA), third-generation packaging vectors pRSV-REV #12253, pMDG.2 #12259, and pMDLg/pRRE #12251 (Addgene, Watertown, MA, USA), and a lentiviral vector directing the expression of luciferase reporter pHIV, Luciferase #21375, at 4.5 µg (Addgene, Watertown, MA, USA). Viral particles were harvested, filtered through a 0.45 µm Durapore PVDF Membrane (Millipore Sigma, St. Louis, MO, USA), and added to each cell line’s culture medium. Viral infections were carried out over 72 h and the transduced cells were selected based on their resistance to 2 μg/mL of puromycin (MP Biomedicals, Santa Ana, CA, USA) [26 (link)].
ID8 (syngeneic) EOC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% heat-inactivated FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1), which were grown under standard conditions. For luciferase transduction, in short, HEK293T/17 (ATCC CRL-11268) cells were plated and co-transfected with Lipofectamine 3000 (L3000015 Invitrogen, Waltham, MA, USA), third-generation packaging vectors pRSV-REV #12253, pMDG.2 #12259, and pMDLg/pRRE #12251 (Addgene, Watertown, MA, USA), and a lentiviral vector directing the expression of luciferase reporter pHIV, Luciferase #21375, at 4.5 µg (Addgene, Watertown, MA, USA). Viral particles were harvested, filtered through a 0.45 µm Durapore PVDF Membrane (Millipore Sigma, St. Louis, MO, USA), and added to each cell line’s culture medium. Viral infections were carried out over 72 h and the transduced cells were selected based on their resistance to 2 μg/mL of puromycin (MP Biomedicals, Santa Ana, CA, USA) [26 (link)].
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