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Confocal Microscopy Image Gallery
Bovine Pulmonary Artery Endothelial Cells (BPAE Line)
P. Del Vecchio established the BPAE cell line in the late 1970s from tissue excised from the main stem of a young cow’s pulmonary artery. The bovine cell line is frequently utilized in studies focusing on conditions such as hypertension, coronary heart disease, and atherosclerosis.
BPAE cells exhibit typical endothelial morphology and are positive for angiotensin converting enzyme, a substance that normally narrows the blood vessels. A class of high-blood pressure medications commonly termed ACE inhibitors target and block the activity of angiotensin converting enzyme. Testing indicates that the BPAE line is also positive for bovine diarrhea virus, a principal viral pathogen of cattle.
The pulmonary arteries carry unoxygenated blood from the heart to the lungs. The walls of the vessels are composed of three layers, the tunica intima (innermost layer), the tunica media (middle layer), and the tunica adventitia (outer layer). The tunica intima is the stratum where endothelial cells are located. These cells are very flat and contain centrally located nuclei. Endothelial cells in vivo are held firmly together by specialized junctions known as tight junctions and gap junctions. The junctions enable endothelial cells to function as selective filters that regulate the passage of molecules and ions across their cell membranes.
GPP130 is a transmembrane protein localized to the cis-Golgi. The culture of bovine pulmonary artery endothelial cells presented in this confocal image was immunofluorescently labeled with anti-GPP130 rabbit monoclonal primary antibodies followed by goat anti-rabbit Fab fragments conjugated to Alexa Fluor 488 (yielding green fluorescence). In addition, the specimen was stained for DNA in the nucleus with the red-absorbing dye TO-PRO-3. Images were recorded with a 60x oil immersion objective using a zoom factor of 4.5 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.
Additional Confocal Images of Bovine Pulmonary Artery Endothelial Cells (BPAE) Cells
Visualizing Intermediate Filaments and the Golgi Complex in BPAE Cells with Immunofluorescence - In a double immunofluorescence experiment, an adherent monolayer culture of BPAE cells was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with a cocktail of mouse anti-vimentin (intermediate filaments) and rabbit anti-GPP130 (Golgi complex) primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 568 (pseudocolored blue) and Alexa Fluor 488, respectively.
Pulmonary Artery Cells Triple Labeled with MitoTracker Red CMXRos, BODIPY FL, and TO-PRO-3 - The culture of BPAE endothelial cells presented in this section was labeled for mitochondria with MitoTracker Red CMXRos and for the cytoskeletal filamentous actin network with BODIPY FL conjugated to phallacidin, a cyclic peptide derived from the toxic death cap mushroom (Amanita phalloides). In addition, the cells were probed for DNA in the cell nucleus with TO-PRO-3 (pseudocolored cyan), a monomeric cyanine nucleic acid stain.
Distribution of F-Actin, Mitochondria, and DNA in BPAE Cell Cultures - The proximity between the mitochondrial and F-actin networks was visualized for this image by treating a culture of bovine pulmonary artery endothelial cells with the mitochondrion-selective stain MitoTracker Red CMXRos and BODIPY FL conjugated to a phallotoxin (phalloidin). The far-red fluorescent DNA probe DRAQ5 was utilized as a nuclear counterstain.
Targeting the Actin Cytoskeleton in Endothelial Cells with Alexa Fluor Probes Conjugated to Phallotoxins - A log phase culture of bovine pulmonary artery cells was labeled with Alexa Fluor 568 (pseudocolored blue) conjugated to phalloidin in order to target the actin cytoskeleton. The cells were also stained with MitoTracker Deep Red 633 and YO-PRO-1, which preferentially bind with intracellular mitochondria and DNA in the cell nucleus, respectively.
BPAE Cells Probed with MitoTracker Orange CMTMRos, BODIPY FL, and DRAQ5 - The triple fluorophore combination of MitoTracker Orange CMTMRos, BODIPY FL conjugated to phallacidin, and DRAQ5 was used to label an adherent log phase culture of BPAE cells for mitochondria, the filamentous actin network, and nuclear DNA, respectively. The cells were first treated with the MitoTracker probe in growth medium for one hour, washed and fixed with paraformaldehyde, permeabilized, and blocked with bovine serum albumen. The cells were subsequently labeled with the conjugated phallacidin and counterstained with the anthraquinone (DRAQ5) reagent.
Filamentous Actin and Mitochondrial Networks in a Culture of Bovine Pulmonary Artery Cells - Details of the mitochondrial and filamentous actin networks were revealed in the cells shown in this confocal image by treating the culture they were resident in with MitoTracker Orange CMTMRos (pseudocolored yellow) and BODIPY FL (pseudocolored blue) conjugated to phallacidin, a mushroom toxin. The culture was also treated with the red-absorbing nuclear counterstain DRAQ5.
Prophase Nucleus in an Isolated BPAE Cell Stained with DRAQ5 - The first stage of mitosis, prophase, can be observed in the image presented in this section. The red-absorbing dye DRAQ5 that was utilized to target the DNA in a culture of BPAE cells reveals that the nucleic acid has condensed into chromosomes in the featured cell. The culture that the cell was resident in was also treated with MitoTracker Orange CMTMRos in order to visualize the mitochondrial network.
Visualizing F-Actin and Mitochondria in Bovine Pulmonary Artery Endothelial Cells - An adherent monolayer culture of bovine pulmonary artery endothelial cells was labeled for the cytoskeletal filamentous actin and intracellular mitochondrial networks with Alexa Fluor 488 conjugated to phalloidin and MitoTracker Red CMXRos, respectively. Cell nuclei were counterstained with a carbocyanine monomer with long-wavelength red fluorescence, TO-PRO-3 (pseudocolored cyan).
BPAE Cells Immunofluorescently Labeled for Nuclear Histone Proteins and Peroxisomes - The log phase culture of BPAE cells presented in the digital image shown in this section was treated with a cocktail of mouse anti-histones (pan) and rabbit anti-PMP 70 (peroxisomal membrane protein) primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies conjugated to Texas Red and Oregon Green, respectively, to target the nuclear histone proteins and peroxisomes.
Triple Stained Endothelial Cells Revealing Cell Nuclei, Mitochondria, and the Actin Cytoskeleton - Three different fluorophores, MitoTracker Red CMXRos, BODIPY FL conjugated to phallacidin, and DRAQ5 were used to label an adherent culture of BPAE cells for mitochondria, the F-actin network, and nuclear DNA. The cells were first treated with the MitoTracker probe in growth medium for one hour, washed and fixed with paraformaldehyde (prepared in growth medium), permeabilized, and blocked with bovine serum albumen. The cells were then labeled with the conjugated phallacidin and counterstained with the anthraquinone (DRAQ5) reagent.
Targeting the Mitochondrial Network and DNA in Cultures of Bovine Pulmonary Artery Endothelial Cells - Details of the mitochondrial network are revealed in this bovine pulmonary artery cell, which was resident in a culture labeled with MitoTracker Red CMXRos, a derivative of X-rosamine. DNA in the nucleus was also targeted with DRAQ5.
BPAE Cells Probed with Alexa Fluor 488, MitoTracker Red CMXRos, and TO-PRO-3 - The culture of BPAE cells presented in this confocal image was labeled for mitochondria with MitoTracker Red CMXRos and for the cytoskeletal filamentous actin network with Alexa Fluor 488 conjugated to phalloidin, a cyclic peptide derived from the toxic death cap mushroom (Amanita phalloides). In addition, the cells were probed for nuclear DNA with TO-PRO-3 (pseudocolored cyan), a monomeric cyanine nucleic acid stain.
Contributing Authors
Nathan S. Claxton, Shannon H. Neaves, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.