Single cell tracking

Franke, K. Adhesion and Single Cell Tracking of Hematopoietic Stem Cells on Extracellular Matrices, Ph.D. Thesis, TU Dresden 2011. 

After harvesting and optional staining, cells are resuspended in chamber medium. Cells are counted and mixed to a final concentration of 4-8 X 10 /ml reporter cells (including intrinsic controls) and 8—16 X 10 /ml consumer cells. In our hands, this concentration is perfectly suited to acquire a moderate cell density that balances the estabhshment of a gradient and also allows single-cell tracking. Naturally, these numbers may not be ideal in assays using different cell types. 

Kasumi A, Sako Y, Yamamoto M (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy. Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J 65 2021-2040... 

Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Letters, 8 (6), 1577-1585. 

Members of the Caliciviridae family can hardly be examined in cell culture or animal models. Therefore, so-called virus-Hke particles (VLP) are employed in current experiments. These particles are expressed recombinantly in insect cells using a baculovirus system and do not carry infectious viral RNA [70-72]. It has been shown by single particle tracking studies that VLPs are internalized into the cells in a similar fashion to native viruses [73]. VLPs are believed to present identical molecular recognition elements to the outside world as do native viruses. 

Single-cell resolution Tracks individual live cells and discriminates them from dead cells and extracellular stain allows identification of hormesis and separation of compensatory adaptation from degenerative change allows more accurate identification of sequence of change in different cytotoxicity parameters as cells might not be synchronous or alike in their response... 

Another technique, single particle tracking, allows one to follow the movement of a single lipid molecule in the plasma membrane on a much shorter time scale. Results from these studies confirm the rapid lateral diffusion within small, discrete regions of the cell sur-... 

Sauer AM, de Bruin KG, Ruthardt N, Mykhaylyk O, Plank C, Brauchle C (2009) Dynamics of magnetic lipoplexes studied by single-particle tracking in living cells. J Control Release 137 136-145... 

Finally, Ruthardt andBraeuchle summarize recent findings, describing transfection pathways of non-viral gene carriers by single particle tracking approaches. This approach allows the detailed identification of potential hurdles for efficient nucleic acid delivery from a single cell viewpoint. 

Interestingly, the diffusional behavior of membrane proteins measured experimentally by FRAP, FCS, or single particle tracking in cells is more complex than predicted by this model. This technique is described best for the case of cell surface proteins, as assessed by FRAP. Such measurements indicate that diffusion is typically much slower than one would expect based on membrane viscosity. In cell membranes, typical values of D for transmembrane proteins are approximately 0.05 pm /s or less, which is much slower than observed in artificial membranes composed of purified lipids. In addition, a significant fraction of proteins is often immobile over the timescale of diffusion experiments (4, 5). Furthermore, diffusional mobilities vary among proteins, and sometimes they differ for the same protein expressed in different cell lines (4, 5). Deviations from pure diffusion are more readily apparent when the trajectories... 

Accumulating evidence clearly points at involvement of the cell cytoskeleton in the compartmentalization of the membrane, in particular, the fine cytoskeleton filaments formed by actin in most eukaryotic cells or spectrin in mammalian red blood cells. However, single-particle tracking experiments show the same patterns of hop-diffusion for lipid molecules located in the extracellular leaflet of the plasma membrane. How can the membrane skeleton, which is located only on the cytoplasmic surface of the membrane, suppress the motion of lipids on the extracellular side ... 

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