Cell separation density gradient

Rreisberg, J.I., Pitts, A.M. and Pretlow, T.G. (1977). Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of Ficoll in tissue culture medium. Am. J. Pathol. 86 591-601. 

Juopperi, T. A., Schuler, W., Yuan, X. et al. 2007. Isolation of bone marrow-derived stem cells using density-gradient separation. Exp Hematol. 35 335-41. 

Each type of blood cell has its own distribution of mass densities (Fig. 2). Most blood cell separators are based on the formation of blood components into layers by density gradient only. Some cell separators, ie, Haemonetics MGS, apply methods based on a combination of mass density and cell size. 

Density Gradient Separation. Based on specific density, each cell in a test tube finds its own position (see Fig. 1), ie, red cells at the bottom, then granulocytes, monocytes, lymphocytes, platelets, and plasma on top. Table 2 Hsts average mass density of the cellular components of blood. The actual numbers vary slightly from person to person. 

In some cases, density gradient solutions are used to separate a specific layer of cells. A solution, like FicoU or PercoU, with a mass density between the density of the cells that are to be collected and the other cells, is added to the blood product. 

Most organelle membranes, such as the tonoplast (6) and the Golgi apparatus (7), can be separated by density gradient ultracentrifugation of plant cell homogenates. However, other effective methods for the isolation of the plasma membrane (8,9) have been described. Moreover, another method that uses an aqueous two-phase system for the isolation of ER is also described (10). Those interested in these details for these methods should consult the original articles. 

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

The diluted blood is carefully layered onto a Ficoll Paque density gradient and centrifuged at 400g for 30min at room temperature without braking to separate the mononuclear cells from erythrocytes and granulocytes. The mononuclear cells at the interface are carefully removed and washed three times in wash medium. 

Now in world practice the most wide-spread is the method of NCs isolation in ficoll density gradient (Rubinstein, et al., 1994). Separation in density gradient enables to obtain predominantly mononuclear cells, but leads to considerable losses of hemopoietic precursors (from 30 to 50%) (Broxmeyer, et al., 1989). 

PF had been proposed as the terminal complex (23) and associated pores were reported on the outer membrane EF (24). Due to their proximity to the site of cellulose ribbon extrusion from the cell surface, these structures were assumed to be responsible for cellulose synthesis. A model was advanced in which cellulose synthase was localized on the outer membrane, which invoked adhesion sites between the outer and plasma membranes as a mechanism to explain the transfer of uridine-diphosphoryl-glucose (UDPG) from the cytoplasm to the cellulose synthases (25,26). However, when the outer and plasma membranes of Acetobacter were isolated separately by density-gradient centrifugation, the cellulose synthase activity was localized only in the plasma membrane fraction (27). Therefore, the linear structures observed on the Acetobacter outer membrane, while they may be associated in some manner with cellulose biosynthesis, are probably not the cellulose synthase terminal complexes. Since no ultrastructural evidence for adhesion sites between the outer and plasma membranes has been presented, a thorough investigation of the mechanism of / (1-4) glucan chain translocation from the cytoplasmic membrane to the outer membrane in Acetobacter xylinvm is now in order. 

For preparation of a density gradient with a large linear range, manufacturers give the following conditions Spin Percoll in 0.15 M NaCl (final concentration) with at least 10 000 x gj,, for Percoll in 0.25 M sucrose apply 25 000 x g. Use near vertical or vertical rotors for cell separation. 

The light scatter signals (FSC and SSC) resulting from flow cytometric analysis of whole blood, lysed whole blood, and a mononuclear cell preparation after the density gradient separation of whole blood are compared in Figure 6.2. Each dot plot shows 2000 cells ... 

The following method will allow researchers to develop the use of density gradient cell separations using spiked huffy coat preparations. Final recovery of carcinoma cells from the huffy coats can be compared with initial known percentages added to samples, allowing refinements of the technique before use with actual samples. 

Cell Harvesting and Cell Suspension Production, 3.2. Density Gradient Separation, and 3.3. Cell Detection Through Immunocy-tochemistry. 

Immunomagnetic cell separation can be used to further debulk re mixture of white blood cells obtained from the density gradient eparation and thus further enrich the suspension with rare cancer ells (see Chapter 21). Before immunochemical staining, it is usual 5 concentrate the cells onto polycarbonate membranes so that they re immobilized (see Note 8). 

It is very important to maintain density gradient and cell suspension at room temperature before and during centrifugation. Temperature affects density and therefore the separation and yield. 

Catsimpoolas N, Griffith AL, Skrabut EM, et al. 1976. Differential Cr uptake of human peripheral lymphocytes separated by density gradient electrophoresis. Cell Immunol 25 317-321. 

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