University of Dundee

Fluorescent reporter protein expression

A popular tool in monitoring gene expression and cellular transfection is the use of fluorescent proteins as reporter molecules. A number of different fluorescent proteins are available displaying an array of excitation and emission spectra. However, not all fluorescence proteins can be accurately measured using all flow cytometers.

The main restriction is the excitation sources available within the instruments. At present CFP, GFP, YFP, dsRed, mCherry, and similar derivatives thereof, are detectable by some or all of the flow cytometers located within the SLS Flow Cytometry Facility. GFP and YFP have very close emission specta, but can be distinbuished using specially selected filter sets on the FACSVerse flow cytometer.

The relatively distinct emission spectra of the fluorescent proteins shown above enables more than one fluorescent protein to be detected in the same cell. A suitable combination for the detection of three different fluorescent proteins in the same sample would be CFP, GFP and mCherry. All three of these proteins are excited by different laser wavelengths (405nm, 488nm and 561nm respectively) making their detection and discrimination relatively trouble free.

By comparing cells expressing the fluorescent protein with cells that do not express the protein, a variety of characteristics can be assessed. For example, the number of cells expressing the protein, the amount of protein expressed, and the heterogeneity of expression.