Steriflip gp filter

To produce virus, hGeCKOa and hGeCKOb plasmid pools (Addgene, #1000000049) were co-transfected into HEK293T cells with lentiviral packaging plasmids pMDLG, pMD2G and pRSV-Rev (Addgene #12251, #12259, #12253). HEK293T cells were cultured in DMEM medium (as described above) and seeded in CellStacks (Corning, CLS3313) the day before transfection. For transfection of one CellStack, 40 μg hGeCKOa+b, 40 μg pMDL.G, 20 μg pMD2.G, and 14.4 μg pRSV-Rev were mixed and diluted with ddH₂O to a total volume of 1,580 μL. 1 mol/L CaCl₂ (Sigma-Aldrich, C7902) was added dropwise to the plasmid-mix during vortexing with low speed. CaCl₂-plasmid-mix was then transferred dropwise to 1,580 μL 2xHBS during vortexing and incubated for 15 minutes at room temperature. Subsequently, 500 mL DMEM medium was mixed with 2,250 μL chloroquine (Sigma-Aldrich, C6628) and the CaCl₂-HBS-plasmid-mix and added to the cells. Medium was exchanged with freshly prepared DMEM medium 6 hours after transfection. Viral particles were harvested after 72 hours later by filtering the viral supernatant through a 0.22-μm Steriflip-GP filter (Sigma-Aldrich, Z660493) and immediately snap-frozen in liquid nitrogen and stored at −80°C until usage.