Representative analysis of transfection efficiencies of rat hepatic stellate cells and myofibroblasts using FuGENE™6 transfection procedure. Rat hepatic stellate cells (rHSCs) were isolated from adult male Sprague-Dawley rats by the pronase-collagenase method and a single-step density gradient centrifugation. Rat myofibroblasts (rMFBs) were prepared by subculturing primary rHSCs by trypsinization at day 7 after seeding following spontaneous activation on a plastic surface. The established murine cell line NIH/3T3 served as a reporter transfection cell line. Cells were transfected with 2 μg of reporter construct pEGFP-C1 and FuGENE™6 according to the suppliers instructions. After incubation for proposed time media were changed and transfection efficiencies were monitored 48 hours after transfection. Representative phase contrast microscopy (A, C, E) and fluorescence microscopy (B, D, F) of rHSCs (A, B), rMFBs (C, D) and NIH/3T3 cells (E, F) are shown. The intensity of fluorescence varies among transfected cells indicating various levels of reporter expression.
Weiskirchen et al. BMC Cell Biology 2000 1:4 doi:10.1186/1471-2121-1-4