Live bovine pulmonary artery endothelial cells (BPAEC) stained with LysoTracker® Red DND-99, dihydrorhodamine 123 and Hoechst 33258.
Live bovine pulmonary artery endothelial cells (BPAEC) were first stained with LysoTracker® Red DND-99 (Cat. no. L7528). Then, a solution of dihydrorhodamine 123 (Cat. no. D632, D23806) and Hoechst 33258 (Cat. no. H1398, H3569, H21491) was added and allowed to incubate with the cells for an additional 10 minutes before the cells were subsequently washed and visualized. The green-fluorescent oxidation product (rhodamine 123, Cat. no. R302) localized primarily to the mitochondria. The red-fluorescent LysoTracker® Red DND-99 stain accumulated in the lysosomes, and the blue-fluorescent Hoechst 33258 dye stained the nuclei. The image was acquired with filters appropriate for DAPI, fluorescein and the Texas Red® dye. The image was deconvolved using Huygens software (Scientific Volume Imaging, http://www.svi.nl/). 3-D reconstruction was performed using Imaris software (Bitplane AG, http://www.bitplane.com/).