X-tremeGENE™ Transfection Reagents General Recommendations

X-tremeGENE™ HP and X-tremeGENE™ 9 Transfection Reagent Comparison

The ability to successfully transfect a cell varies significantly depending on the cell type, protocol, and the composition of the transfection reagent. X-tremeGENE™ transfection reagents are designed to efficiently transfect common and difficult-to-transfect cells with a variety of molecules, including DNA, small RNAs, and CRISPR/Cas9 components.

Primary fibroblasts were isolated from human foreskin and transfected with 4 μL X-tremeGENE^™ HP Reagent and 1 μg of GFP-encoding plasmid DNA in a 6-well cell culture plate. GFP expression was visualized 48 hours after transfection. The left panel shows GFP fluorescence, and the right panel shows the corresponding brightfield image.

Human mesenchymal stem cells were isolated from bone marrow aspirate of four different donors. Cells were transfected using X-tremeGENE^™ HP Reagent (XHP), X-tremeGENE^™ 9 Reagent (X9), and a competitor reagent (LTX), using the indicated ratios (μL reagent : μg GFP-encoding plasmid DNA. Ratio 2:1 was not tested using X-tremeGENE^™ 9 Reagent). Untransfected cells served as control (neg). FACS analysis was performed 48 hours after transfection.

Cells were transfected using X-tremeGENE^™ HP Reagent (XHP), and competitor reagents (LTX and L2K), at the indicated ratios (μL reagent : μg GFP-encoding plasmid DNA). Transfection efficiency was visualized using GFP fluorescence microscopy.

Note: The above data was produced using either a GFP-encoding pcDNA3.1 plasmid or a luciferase-encoding pCI plasmid, both with the cytomegalovirus (CMV) promoter. These recommendations are guidelines based on experimental findings. The optimal reagent:DNA ratio must be determined empirically.