Simultaneous Analysis of GFP and CELL CYCLE

Requests for the GFP-Spectrin plasmids should be addressed to:
Robert F. Kalejta,
RKalejta@molbio.princeton.edu
609-258-5993
 

Requests for the Us9-GFP plasmid (pBB14) should be addressed to:
Prof. L. Enquist
Lenquist@molbio.princeton.edu
609-258-2415
 
 

We have developed a method for quantification of GFP as a marker of transfected cells and cell cycle analysis by flow cytometry. Cell cycle position is determined by DNA content as measured by propidium iodide (PI) staining. For optimal PI staining, cells require permeabilization with ethanol to allow the dye access to the nucleus. However, such treatment with ethanol results in the loss of GFP fluorescence as the soluble, cytoplasmic GFP leaks out of the cells following permeabilization. The use of fixatives (e.g. paraformaldehyde) that retain GFP in the cells after ethanol permeabilization result in poor staining of the DNA with PI. To circumvent this problem, we developed a simple assay employing a membrane-localized or a transmembrane GFP fusion protein that is retained in cells following ethanol permeabilization facilitating the simultaneous detection of GFP (transfection marker) and high-resolution PI data (cell cycle analysis).
[Cytometry, 29:286-291 (1997), Kalejta RF, Shenk T and Beavis AJ. "Use of a Membrane-Localized Green Fluorescent Protein Allows Simultaneous Analysis of Transfected Cells and Cell Cycle Analysis by Flow Cytometry".
Exp. Cell Res. 248:322-328 (1999), Kalejta RF, Brideau AD, Banfield BW and Beavis AJ.
"An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry".]

Initially, we used the membrane-localized GFP-fusion protein GFP-Spectrin. The GFP-Spectrin localizes to the pleckstrin hmology domain of the inner leaflet of the plasma membrane (Wang et al, 1996) and is retained during ethanol permeabilization, permitting the simultaneous detection of GFP-expressing, transfected cells. Recently, we introduced an improved fusion protein, Us9-GFP (Brideau et al, J. Virol, 72, 4560-4570. 1998) which is a transmembrane fusion protein and is retained quantitatively in cells following ethanol permeabilization. Therefore, since there is no loss of Us9-GFP fluorescence, this permits maximal detection and resolution of transfected cells, particularly those cells expressing low levels of GFP.
 

Effects of Ethanol Permeabilization on Expression of Cytosolic EGFP or Two Novel GFP-Fusion Proteins, GFP-Spectrin and US9-GFP

Dual-colour, flow cytometric analysis was used to assess the effects of the expression of certain cell cycle regulators co-expressed with GFP-spectrin or Us9-GFP in transient transfection assays.
 

Two-Colour Immunofluorescence Analysis of Transfected Cells Using Us9-GFP and Propidium Iodide 

The GFP(+) cells [shown in green] are clearly resolved from the GFP(-) cells [shown in red]. The subpopulations can be gated onto a single parameter histogram of PI fluorescence where the relative percentages of cells in each phase of the cell cycle can be quanititated.

References:

Cytometry, 29:286-291 (1997), Kalejta RF, Shenk T and Beavis AJ.
"Use of a Membrane-Localized Green Fluorescent Protein Allows Simultaneous Analysis of Transfected Cells and Cell Cycle Analysis by Flow Cytometry".

Exp. Cell Res. 248:322-328 (1999), Kalejta RF, Brideau AD, Banfield BW and Beavis AJ.
"An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry".