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Confocal Microscopy Image Gallery
Human Bone Osteosarcoma Cells (U-2 OS Line)
J. Ponten and E. Saksela established the U-2 OS human cell line from a sarcoma of the tibia that was moderately differentiated. Males are more commonly afflicted with osteosarcoma than females, but the patient from whom the original cells were sampled was a fifteen-year-old girl.
U-2 OS cells exhibit epithelial characteristics and are positive for both insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF II) receptors. The cells express a number of antigens, including blood type A, Rh+, HLA A2, Aw30, B12, Bw35, and B40(+/-), and have been found to be negative for simian virus 40 (SV40), respiratory syncytial virus (RSV), and adenoviruses. In 1972, it was discovered that major U-2 OS stocks were contaminated with mycoplasmata, but the microorganisms were subsequently eradicated from the line.
Osteosarcoma is a cancer that most commonly affects adolescents. This is thought to be because the cells in which this type of tumor originates, the osteoblasts, are most active during periods of rapid growth. Similarly, since boys tend to grow more than girls, they are significantly more likely to develop osteosarcoma than members of the opposite sex. The tumors typically form in the large bones of the legs or arms, but other sites are also possible. Signs of osteosarcoma vary somewhat, but often include swelling, pain, and limited movement of the affected limb.
In order to visualize the F-actin and mitochondrial networks in a culture of human osteosarcoma cells, the culture was treated with BODIPY FL conjugated to phallacidin and MitoTracker Red CMXRos. The nuclei in the cells were counterstained with TO-PRO-3, a carbocyanine monomer with long-wavelength red fluorescence. Images were recorded with a 60x oil immersion objective using a zoom factor of 2.0 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.
Additional Confocal Images of Human Bone Osteosarcoma (U-2 OS) Cells
Immunofluorescently Targeting the Microtubule Network in Human Osteosarcoma Cells - Immunofluorescence with mouse anti-alpha-tubulin was employed to visualize the details of the microtubule network in a log phase monolayer culture of human osteosarcoma cells. The secondary antibody (goat anti-mouse IgG) was conjugated to Alexa Fluor 488. In addition, mitochondria in the cells were targeted with MitoTracker Red CMXRos, a derivative of X-rosamine.
U-2 OS Cells Triple Labeled with BODIPY FL, MitoTracker Red CMXRos, and TO-PRO-3 - The culture of U-2 OS cells presented in this section was labeled with MitoTracker Red CMXRos and BODIPY FL conjugated to phallacidin, targeting the mitochondrial network and filamentous actin, respectively. The culture was counterstained for DNA in the cell nucleus with TO-PRO-3.
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.