D-Lin-MC3-DMA, an ionizable cationic lipid, is a potent siRNA delivery vehicle.

Replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro.

White leghorn chicken embryos at stage 24 (30) were removed and dissected in Hanks buffered saline solution (HBSS).?Forelimbs were pooled and treated for 1 h in dispase to remove epithelial ectoderm.?Limb mesenchyme was dissociated, and resuspended in DMEM with 5% FBS and 1% penicillin/streptomycin.?Cells were plated onto 96-well plates at a density of 2x10^5 or 2x10^7 cells/ml and treated with 0-40 μg/ml LNP-pDNA.?Cultures were incubated overnight at 37℃ and resuspended in PBS buffer for expression analysis using a BD LSRII flow cytometer.?Cell survival was assessed in low-density cultures after 24 h of treatment, by counting the adherent cells remaining after PBS washes.?Viability was determined based on average counts of all adherent cells within a single field of view (100x) normalized to cell counts of untreated cultures.