Fluorescence produced by transfection reagents can be confused with green fluorescent proteins in mammalian cells.
The Aequorea victoria green fluorescent protein (GFP) reporter system is a convenient way to monitor gene expression and other cellular functions in mammalian cells. To study gene expression, a GFP-fusion plasmid construct is often transfected into mammalian cells using a variety of methods including calcium phosphate- and liposome-based DNA transfer. Subsequently, the expression of GFP-fusion protein is monitored by fluorescence microscopy or flow cytometry. Here, we report that certain transfection reagents can produce fluorescence that can be detected in a wide range of wavelengths, which can be confused with GFP-fusion protein. The fluorescence false positives can be a problem, particularly when the GFP expression levels are low. To improve the GFP-based detection or screening methods, it is imperative to include an appropriate negative control and to detect GFP using a narrow-wavelength emission filter corresponding to the emission spectrum around the GFP peak.
Guo
B
B
Northeastern Ontario Regional Cancer Centre, 41 Ramsey Lake Road, Sudbury, ON, Canada P3E 5J1.
Pearce
A G
AG
Traulsen
K E
KE
Rintala
A C
AC
Lee
H
H
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D008322:Mammals; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards; D010957:Plasmids; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods* / Q000592:standards
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins