Analysis of cell-cycle profiles in transfected cells using a membrane-targeted GFP.

At present, the most commonly used method for determining the cell-cycle profiles (G1, S and G2/M phases) in mammalian cells is to co-transfect the gene(s) of interest with a second plas-mid expressing a cell-surface protein that can be identified by immunofluo-rescence-based cell staining and then analyze the fluorescently stained cells using flow cytometry (9,10). The cluster of differentiation (CD) proteins of T and B lymphocytes are often chosen as the cell-surface markers for this purpose because they are only expressed in T and B cells and, even when expressed at high levels, are not toxic to the common recipient cells used in transfection assays (9,10). However, detection of the cell-surface marker in these trans-fection assays requires a complicated protocol and reagents. The cells must be detached from tissue culture plates without trypsinization because the cell-surface marker can be destroyed by trypsin. Such treatment, however, makes it difficult to generate a single-cell suspension that is essential for flow cytometry analysis. After detachment from the plates, the cells have to be stained with a specific monoclonal an-tibody against the transfected cell-surface marker. To detect the signal in flow cytometry analysis, the monoclon-al antibody used in the assay needs to be either directly conjugated with fluor-escein isothiocyanate (FITC) or detected by FITC-conjugated anti-mouse