DNA (pGFP which expresses green fluorescent protein) was transfected into the cell types indicated using Attractene Reagent according to the conditions recommended in the handbook. Transfection was also performed with [A] Reagent F using 2 reagent volumes recommended by the manufacturer or [B] Reagent L according to the manufacturer's recommendations, with and without the enhancer provided. Transfection efficiency was estimated by counting the number of fluorescent cells by FACS analysis. Transfection efficiency is expressed relative to efficiency using Attractene Reagent which was set at 100%.
HepG2 cells were transfected with DNA (pGFP) using Attractene Transfection Reagent or Reagent L from another supplier according to the manufacturer's instructions. FACS analysis confirmed equal numbers of transfected cells in both cultures. Two days after transfection, cells were examined by light microscopy. [A] Cells transfected using Attractene Reagent were healthy and viable. [B] In contrast, cells transfected using Reagent L displayed high levels of cell death. [C] Cell viability was measured using a CellTiter-Blue assay (Promega). Viability was significantly lower in cells transfected using Reagent L compared to cells transfected using Attractene Reagent (set at 100%).
Traditionally, cells are seeded the day before transfection. Using the Fast-Forward Protocol, seeding and transfection take place on the same day. This saves time and effort.
HEK293 cells were [A] transfected with a plasmid expressing green fluorescent protein (pGFP) only, or [B] cotransfected with pGFP and a plasmid expressing an shRNA targeted against the green fluorescent protein gene (pGFP + pshGFP), or [C] cotransfected with pGFP and a negative control plasmid expressing a scrambled shRNA (pGFP + pNegative). After cotransfection of pGFP and the shRNA vector pshGFP, the green fluorescent protein was effectively silenced indicating effective cotransfection.
