Plvx tre3g

Half a million HEK293T cells were seeded per well in a 6-well plate (one plate per lentivirus) in 2 ml of High glucose DMEM (Life Technologies, 11995) with 10% FBS (Sigma, F2442). The next evening the medium was replaced with fresh DMEM and cells were transfected with 100 μl of transfection mixture per well. The transfection mixture contained 3 μl X-treme Gene 9 reagent (Roche, 06365787001), 500 ng psPAX2 (psPAX2 was a gift from Didier Trono, Addgene plasmid # 12260), 50 ng pMD2.G (pMD2.G was a gift from Didier Trono, Addgene plasmid # 12259), 500 ng of pLVX-Tet3G (Clontech) or pLVX-TRE3G-Luciferase (Clontech) or pLVX-TRE3G-LbNOX or pLVX-TRE3G-mitoLbNOX or pLVX-TRE3G-TPNOX or pLVX-TRE3G-mitoTPNOX and Opti-MEM medium (Life Technologies, 31985-070) up to 100 μl. To make the transfection mixture, 50 μl solutions of X-treme Gene 9 and DNA mixtures were prepared separately and the DNA solution was added dropwise to the X-treme Gene 9 solution. The mixture was incubated at room temperature for 30 min before adding it to cells. Two days after transfection, medium was collected, centrifuged at 500 x g for 5 min to pellet cells and the supernatant was aliquoted and stored at −80 °C.