An ultracentrifuge based on the air turbine of a dental drill attains 400,000–600,000 rpm and a centrifugal force 2 to 3 million ×g. It is used for the examination of cell suspentions and homogenates and can be applied to macromolecular solutions. Microcuvettes with volumes ranging from less than 1 mm3 to 0.001 mm3 (suitable for single cells) are of disk and square capillary form and are made of polycarbonate or glass. A special microscope is used to obtain a stopped image of the rotating cuvette. A new method to preserve the centrifugal state of the contents is tested: deep freezing of a rotating cuvette for subsequent freeze-drying and microscopical examination. Experiments on cells include centrifugal intercellular movement of nuclei and cytoplasmic particles, cell fusion, and homogenization.
Beta-2 microglobulin (B2M) is an immune system protein that is found on the surface of all nucleated human cells. B2M is naturally shed from cell surfaces into the plasma, followed by renal excretion. In patients with impaired renal function, B2M will accumulate in organs and tissues leading to significantly reduced life expectancy and quality of life. While current hemodialysis methods have been successful in managing electrolyte as well as small and large molecule disturbances arising in chronic renal failure, they have shown only modest success in managing plasma levels of B2M and similar sized proteins, while sparing important proteins such as albumin. We describe a systematic protein design effort aimed at adding the ability to selectively remove specific, undesired waste proteins such as B2M from the plasma of chronic renal failure patients. A novel nanoparticle built using a tetrahedral protein assembly as a scaffold that presents 12 copies of a B2M-binding nanobody is described. The designed nanoparticle binds specifically to B2M through protein–protein interactions with nanomolar binding affinity (~4.2 nM). Notably, binding to the nanoparticle increases the effective size of B2M by over 50-fold, offering a potential selective avenue for separation based on size. We present data to support the potential utility of such a nanoparticle for removing B2M from plasma by either size-based filtration or by polyvalent binding to a stationary matrix under blood flow conditions. Such applications could address current shortcomings in the management of problematic mid-sized proteins in chronic renal failure patients.
Centrifugation of celt suspensions containing two or several cell types at forces up to 2 million g results in several basic events in succession: 1. Intracellular stratification. 2. Extrusion of the most dense cell parts (nuclei or cytoplasmic components) and their penetration into an adjacent cell in the compact sediment. Such introduction of protoplasmic elements from one cell into another is considered as centrifugal hybridization and fusion of cells. It differs from other methods of cell hybridization by its selectivity for cell components. 3. Further intermingling and mixing of ceils into a fused protoplasmic mass. 4. With continuing increase of centrifugal force fractions of subeellular components are formed from the protoplasmic maSS. These components are presumably viable since eells are not exposed to chemical treatment. Morphological demonstration of hybridization is hased on centrifuging microscopy and on labeling donor cells or recipient cells by staining or fluorescence. Genetic evidence can be provided by cultivation of hybrid cells in vitro and their cloning in vivo.
SUMMARY Microscopy of cells growing in vessels containing plastic films as a substrate or as a transparent window is facilitated by a contact cap on the objective or the contact objectives for intravital microscopy. When applied to microscopical examination of living cells through a thin film, the cap considerably improves the conditions of observation with high‐power dry objectives and makes it possible to use water‐ and oil‐immersion objectives.
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