Separation density gradient

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. 

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 ... 

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

Variations in Properties of Coal Macerals Elucidated by Density Gradient Separation... 

All density gradient separations are carried out in aqueous cesium chloride solutions to which a nonionic surfactant has been added (polyoxyethylene-23-lauryl ether, Brij-35). The surfactant is necessary to disperse the fine coal particles in the aqueous medium. Without complete particle dispersion the separation will most likely not be optimal. 

Preparative II DGC in Figure 1 is a scaled-up version of Preparative I which uses a combination of the sink-float separations followed by non-linear density gradient separation. With this technique 20-25 g of a maceral concentrate can be separated. In effect, for a three-fold increase in time for separation, isolation, and characterization, we obtain at least a ten-fold increase in the yield of maceral materials. 

The isopycnic method has been used to dramatically demonstrate semiconservative DNA replication, using CsCl density gradients. Separation of DNA, RNA, protein, and carbohydrates can be performed in dense CsCl solutions, where the RNA pellets, the DNA forms bands, and protein and carbohydrates form a thin layer called a pellicle at the top of the gradient. 

Peripheral blood mononuclear cells were obtained by density gradient separation through Ficol-Hypaque technology 17). Thymidine incorporation proliferation assays were set up as previously described using human serum albumin (HSA) or STn-HSA as stimulant (77). A stimulation index of > 2 SD compared to normal donors mononuclear cells was used as evidence of positive response. Fifty seven percent of tested vaccinated patients had evidence of specific T cell proliferation against STn. 

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

Figure 4. Alfalfa somatic embryo proteins separated after extraction by sucrose density gradient separation (left side of chromatogram) and SDS-polyacrylamide electrophoresis (right side of chromatogram). The 2,4-D-treated embryos express more IIS protein as shown in the sucrose gradient. The IIS protein has polypeptides that co-migrate with IIS seed protein as shown by SDS-PAGE. (Reproduced with permission from reference 4. Copyright 1985, Plenum Publishing.)...

The general principles and some examples of density-gradient separation have been discussed by Charlwood (1966). The technique of using sucrose gradients (Pickels, 1943) to stabilize weak sedimenting boundaries has been furthered by Brakke (1951, 1953) and others. 

Density-gradient separations of cytoplasmic fractions from liver have been carried out to study the distribution of activity of several enzymes (Thomson, 1959). Ribosomes and polysomes have been fractionated to elucidate the mechanisms of protein synthesis (Watson, 1964), Various viruses have been studied with the density-gradient technique polio virus (Levintow and Darnell, 1960), Rous sarcoma virus (Crawford, 1960), Shope papilloma virus (Williams et ai, 1960), and adenoviruses (Allison et ai, 1960). Density-gradient fractionation has been particularly useful for separation of DNA molecules from various species of bacteria (Rolfe and Meselson, 1959) and from animal cells (Kit, 1961), Other types of molecules, e.g., antibodies, lipoproteins, and rheumatoid factor have been isolated by density-gradient methods [see Charlwood (1966) for examples]. 

Dobrota M. and Hinton R. 1992. Conditions for density gradient separations. In D. Rickwood (Ed.), Preparative Centrifugation A Practical Approach, p. 77. New York, Oxford University Press. 

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