GFP transfection Efficiency Assay

Validate transfection success using the NucleoCounter® NC-3000™

  • Fixed protocol – no settings needed
  • Fully automated – just load sample and press run
  • High specificity – assess transfection at the single cell level
  • Visualize results in images, scatter plots and GFP histograms
  • Obtain precise and reproducible results

Overview of the GFP transfection efficiency assay

The introduction and expression of foreign genes in eukaryotic cells is a technique widely used for investigating gene regulation and function. It is important that scientists performing these techniques know what proportion of cells have taken up this new gene, a measurement known as the transfection efficiency. This is a critical measure to have, as it greatly affects process optimization and interpretation of results down-stream.

A simple method to determine transfection efficiency is by using Green Fluorescent Protein (GFP) as a reporter. Using an appropriate promoter, GFP can be expressed in the cells by itself or attached to the protein of interest as a fusion protein. With a GFP reporter system, the transfection efficiency can easily be determined as the percentage of cells expressing GFP in the entire cell population.

NucleoCounter® NC-3000™ is equipped with a user-friendly GFP-transfection efficiency assay and uses a very low sample volume. Sample preparation for GFP transfection efficiency with the NucleoCounter® NC-3000™ is quick and easy. Just add solutions 15 and 16 to your sample, incubate at 37˚C and load your sample onto an NC-Slide A2™; for high throughput, use an NC-Slide A8™. Within minutes, you can view images and corresponding data presented in scatter plots and histograms for up to eight samples.

Assay principle

Principle of the plug and play GFP Transfection Efficiency Assay using the NucleoCounter® NC-3000™:


  • Cells are located using Hoechst 33342 (blue) and the percentage of GFP expressing cells (green) can easily be determined. Non-viable cells are stained with propidium iodide (PI; red)
  • In the accompanying NucleoView™ software, all cells stained with Hoechst 33342 appear blue
Cell count visualized in the NucleoView™ software. GFP-expressing cells are green, non-viable cells are red and viable cells are blue.


Image of processed data from the GFP Transfection Efficiency Assay in the NucleoView™ software, in the form of scatter plots.
Images acquired from the NucleoCounter® NC-3000™ advanced image cytometer’s GFP Transfection Efficiency Assay are quantified using their corresponding scatterplot.

Using the NucleoView™ software, you may identify and select populations and/or single cells in the fluorescent data images to see where they lie on the corresponding scatterplots. This allows full traceability of GFP and PI intensities for single cells and allows you to precisely identify cells for analysis and visually confirm their staining status. Therefore, you can set distinct gates between GFP+ and GFP- cells, as well as differentiate live and dead cells with confidence.

This two-tier process of data generation and confirmation is an advantage that comes with using the NucleoCounter® NC-3000™ advanced image cytometer. Within the NucleoView™ software, you can create and save your gates as templates and protocols, allowing you to apply them to future runs, minimizing analysis time.