NC-3000™ GFP Transfection Efficiency Assay
For easy, fast and objective measurement of transfection efficacy
TOTAL ASSAY TIME LESS THAN ONE MINUTE!
Introduction and expression of foreign DNA into an eukaryotic cell (transfection) is widely used for studying gene regulation and function. It is often required to determine the transfection effi ciency either for optimising transfection conditions or because the down-stream experimental set-up relies upon accurate determination of transfection ratio.
An easy way to determine the transfection effi ciency is to use the gene encoding Green Fluorescent Protein (GFP) as a transfection reporter. The vector harbouring the gene of interest can be constructed so it also carries GFP either as a fusion or under control of its own promoter. Such a construct enables determination of transfection effi ciency by simply determining the ratio of cells expressing GFP to the total cell number. The NucleoCounter® NC-3000™ enables two assays for measuring GFP transfection efficiency; a fast method taking less than a minute and an advanced assay which may be used for weak GFP expression and which also provides information about the transfection ratio of nonviable cells.
Read more
Principle
In order to determine the transfection ratio, a suspension of cells transfected with GFP is stained with either VB-48 (the fast method) or Hoechst and PI (the advanced method). After staining cells are loaded into either of two types of ChemoMetec slides: the 2-chamber NC-Slide A2™ or the 8-chamber NC-Slide A8™. Samples are analyzed using the NucleoCounter® NC-3000™ system. For both methods the transfection ratio and a histogram showing the GFP intensity is shown, moreover, for the advanced method the transfection ratio of nonviable cells are also determined.
24 hours post transfection with a GFP harbouring plasmid, cells were harvested, stained with Solution 15 (Hoechst) and Solution 16 (PI) and analysed using NC-3000. A histogram (upper right) shows the fluorescence intensity of viable GFP transfected cells. The image of the transfected cells acquired by the NC-3000 is shown below. Cells appearing green are GFP expressing cells, cells appearing red are nonviable cells (PI permeable cells), while cells appearing blue are nontransfected, viable cells. A result box shows information (advanced assay) about transfection ratio, viability and GFP intensity.
Key benefits
- Fast and easy-to-use assay for measuring transfection efficiency
- User friendly protocol with predefined settings
- Fully automated analysis procedure
- GFP intensity histogram displayed
- Objective analysis – standardized results
- Total assay time less than one minute