 |
 |
 |
 |
||
|
 |
|||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
 | ||||||||||||||||||||||||
Confocal Microscopy Image Gallery
Male Rat Kangaroo Kidney Epithelial Cells (PtK2 Line)
PtK2 cells are commonly utilized as a model of the mitotic process because the cells and the fourteen chromosomes they each contain are quite large compared to the chromosomes of humans and other animals. The line was established from renal tissue excised from a male Potorous tridactylus, popularly known as the rat kangaroo.
PtK2 cells exhibit typical epithelial characteristics and stain positive for the keratin, an intermediate filament protein. Studies indicate that the cells are susceptible to infection with several different viruses, including herpes simplex, coxsackievirus A9, vesicular stomatitis (Ogden strain), and vaccinia. The line demonstrates resistance against poliovirus 2, adenovirus 5, and coxsackievirus B5.
In addition to their size, PtK2 cells are well suited for chromosome studies because under the proper conditions they stay relatively flat during mitosis, making it easier to clearly observe the process under a microscope. A line of female rat kangaroo kidney epithelial cells, called PtK1, exhibits similar favorable characteristics as the PtK2 line, but the cells contain an even smaller number of chromosomes, so that they also are popular for visualizing mitosis. When maintained at 37 degrees Celsius, PtK2 cells can complete all the stages of cellular division in two to three-and-a-half hours. Cultures of the cells that are healthy and growing frequently contain some cells in each stage of mitosis.
A log phase culture of PtK2 epithelial cells was labeled with MitoTracker Red CMXRos to target the mitochondrial network and Alexa Fluor 488 conjugated to phalloidin, which binds to the filamentous actin cytoskeleton. In addition, the cells were treated with TO-PRO-3, which exhibits affinity for DNA, in order to counterstain the nucleus. Images were recorded with a 60x oil immersion objective using a zoom factor of 3.5 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, the Alexa Fluor 488 image channel was pseudocolored blue, while the MitoTracker Red CMXRos image channel was pseudocolored red and the TO-PRO-3 channel was pseudocolored green.
Additional Confocal Images of Male Rat Kangaroo Kidney Epithelial (PtK2) Cells
Employing Phallotoxins to Target the F-Actin Network in Monolayer PtK2 Cell Cultures - Filamentous actin was targeted in the culture of rat kangaroo kidney (PtK2) cells presented in this section with BODIPY FL conjugated to phallacidin, a bicyclic peptide derived from a toxic mushroom. In addition, the cells were treated with the mitochondrion-selective stain MitoTracker Red CMXRos and a nuclear counterstain, TO-PRO-3 (pseudocolored cyan).
Rat Kangaroo Kidney Epithelial Cells with MitoTracker Red CMXRos, BODIPY FL, and DRAQ5 - An adherent monolayer culture of male rat kangaroo kidney epithelial cells was treated with MitoTracker Red CMXRos in growth medium for one hour, washed, and fixed with 3.7-percent paraformaldehyde in medium containing serum. After washing and permeabilization, the cells were blocked with bovine serum albumen in PBS and labeled with BODIPY FL conjugated to phallacidin. The nuclei were subsequently counterstained with DRAQ5 (pseudocolored cyan), an anthraquinone that exhibits preferential intercalation at AT base pairs.
Enhanced Green Fluorescent Protein in Transfected PtK2 Cells - The PtK2 epithelial cell culture illustrated in this section was transfected with a chimeric EGFP plasmid vector that expresses a fluorescent fusion protein targeted at keratin intermediate filament networks. The specimen was also stained for mitochondria with MitoTracker Red CMXRos, and for the nucleus with the DNA-selective compound Hoechst 33342 (pseudocolored cyan).
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.