 |
 |
 |
 |
||
|
 |
|||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
Confocal Microscopy Image Gallery
Fox Lung Fibroblast Cells (FoLu Line)
Established from the lung tissue of an adult female grey fox (Urocyon cinereoargenteus), FoLu cells exhibit many of the typical characteristics associated with fibroblasts and are primarily used in virus studies. Many lung-derived cell lines similar to FoLu have also been utilized in studies focusing upon the short- and long-term effects of cigarette smoke, and this particular line is potentially useful for such research as well.
Testing has demonstrated that the FoLu line is susceptible to vesicular stomatitis, herpes simplex, and vaccinia viruses, but is resistant to poliovirus. The cells are negative for reverse transcriptase, indicating a lack of integral retrovirus genomes. Their growth in culture is adherent, the cells generally forming monolayers that adhere to glass or plastic surfaces.
Traditionally scientists viewed fibroblasts in relatively narrow terms, often considering them to be akin to scaffolding for more important cells. However, most modern studies suggest that many of these cells, which are primarily found in connective tissue, can function in very specialized and important ways. According to recent research, depending on the specific surface markers they possess, most fibroblasts can transform into either scar-producing myofibroblasts or into a variety of fat cells known as lipofibroblasts. A group of scientists at the University of Rochester Medical Center have identified the protein Thy-1 as a key marker of a fibroblast cell's potential for development, those fibroblasts that express the protein apparently possessing the capability of becoming myofibroblasts while those with no Thy-1 have the potential to become lipofibroblasts.
A monolayer culture of fox lung cells (shown above) was treated with MitoTracker Red CMXRos in medium containing 15 percent Cosmic calf serum, fixed with the same medium containing 3.7 percent paraformaldehyde, and permeabilized with 0.2 percent Triton X-100. The cells were then labeled for the filamentous actin network with Alexa Fluor 488 (pseudocolored blue) conjugated to phalloidin and counterstained with TO-PRO-3 (pseudocolored green), targeting DNA in the nucleus. Images were recorded with a 60x oil immersion objective using a zoom factor of 2.5 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser, the 543-nanometer line from a green helium-neon 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 unless otherwise noted above.
Additional Confocal Images of Fox Lung Fibroblast (FoLu) Cells
FoLu Cells Labeled for F-Actin with an Alexa Fluor Probe Conjugated to a Phallotoxin - The culture of FoLu fibroblasts presented in this section 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 DNA in the cell nucleus with TO-PRO-3, a monomeric cyanine nucleic acid stain.
Visualizing the Proximity Between the Nucleus and the Mitochondrial Network in Fox Lung Cell Cultures - Details of the mitochondrial network in fox lung fibroblast cells were revealed by treating a FoLu culture with MitoTracker Red CMXRos, a popular red-fluorescent mitochondrion-selective stain. The high-affinity DNA probe DRAQ5 (pseudocolored cyan) was utilized to counterstain cell nuclei in the culture.
F-Actin, Mitochondria, and DNA Distribution in FoLu Fibroblasts - A log phase culture of fox lung fibroblast cells was labeled for F-actin and the intracellular mitochondrial network with Alexa Fluor 488 conjugated to phalloidin and MitoTracker Red CMXRos, respectively. DRAQ5 (pseudocolored cyan), a far-red fluorescent DNA probe, was employed as a nuclear counterstain.
Targeting Nuclear Pore Complexes in Fox Lung Fibroblast Cells with Immunofluorescence - Nuclei in a culture of FoLu cells were stained with Hoechst 33342 and immunofluorescence was utilized to visualize the numerous nuclear pore complexes located on each nucleus. The cells were fixed, permeabilized, blocked with 10-percent normal goat serum, and treated with mouse anti-NPCP (nuclear pore complex protein) primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 647 (pseudocolored yellow). The mitochondrial network was also targeted by treating the fibroblasts with MitoTracker Red CMXRos.
Intermediate Filaments in FoLu Fox Lung Fibroblasts - In order to label the intermediate filaments in the log phase adherent FoLu culture shown in this confocal image, the fixed and permeabilized cells were blocked and treated with mouse anti-vimentin (porcine eye lens) primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 488. Cell nuclei were counterstained with the red-absorbing dye TO-PRO-3.
Details of the Mitochondrial Network in Fox Lung Cells Revealed with a MitoTracker Probe - MitoTracker Red CMXRos is a derivative of X-rosamine that selectively binds with mitochondria. By treating a culture of fox lung cells with the MitoTracker probe, details of the mitochondrial network were visualized. The fibroblasts were also stained with DRAQ5 (pseudocolored cyan), a far-red fluorescent DNA probe, in order to image cell nuclei.
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.