ECV304 cells lines constitutively expressing the cytoplasmic (cyto-roGFP) and mitochondrial (mito-roGFP) form of roGFP.

<p>Cells were grown in glass chamber slides at concentrations to allow 50–70% confluence in 24 hrs. On the day of experiments, cells were washed with PBS three times, counterstained with the mitochondrial marker MitoTracker Red and the nuclear marker <b><i>Hoechst</i></b>, fixed with 4% paraformaldehyde and mounted for fluorescence microscopy visualization. Images (A) and (C) depict respectively merged photos of ECV304 cells expressing the cyto-and mito-<i>roGFP</i> (green) protein, <b><i>Hoechst</i></b> staining (blue) and bright-field (40X, NA = 1.00). Images (B) and (D) depict respectively merged photos of ECV304 cells expressing the cyto-and mito-<i>roGFP</i> (green) protein, counterstained with <b><i>Hoechst</i></b> (blue) (100X, NA = 1.35)). The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048817#pone-0048817-g005" target="_blank">figure 5F</a>, depicts the merged photo of ECV304 cells expressing the mito-roGFP protein (D) and ECV304 cells stained with the mitochondrial marker MitoTracker Red (E). (100X, NA = 1,35)).