Erythrocytes density gradient centrifugation

Fig. 6.2. The FSC and SSC signals resulting from the cells in different blood preparations (whole peripheral blood whole peripheral blood after erythrocyte lysis and peripheral blood mononuclear cells [PBMCs] with granulocytes removed by density gradient centrifugation). The bottom panels indicate the five clusters into which the scatter signals fall.

Enrichment of rare cancer cells from peripheral blood samples is an application that typically requires density gradient centrifugation as a first step. Application of this technique addresses two objectives depletion of erythrocytes and depletion of polymorphonuclear cells. It is expected that cancer cells undergo sedimentation with the mononuclear cell fraction because of their similar density. However, some studies have found that cancer cells are also lost in the polymorphonuclear fraction or the erythrocyte fraction (8,9). Optimization of the density gradient sedimentation step is an important issue in such an application, because it will determine the recovery of rare cells from blood and affect the chances of their detection by immunochemical means. 

Remove erythrocytes from the sample by density gradient centrifugation, as described in Chapter 38. Aliquot approx 10 cells into each labeled sample tube. 

In the cell cultures neutrophils in RPMI-1640 medium were used. They were isolated from human blood by density gradient centrifugation and erythrocyte lysis. We used phor-bol 12-myristate 13-acetate to stimulate them by exchanging the medium in the wells. This chemical stimulant mimics the presence of a pathogen which leads to the chromatin release. The control culture did not receive the stimulating agent and was kept unstimulated. 

Mixed leukocytes are prepared using dextran sedimentation of erythrocytes, centrifugation of leukocytes from the plasma, and lysis of residual erythrocytes with ammonium chloride. Granulocytes and mononuclear leukocytes are separated from each other and from erythrocytes by density gradient centrifugation using a mixture of Ficoll, Hypaque and dextran. Thirty ml of blood provides sufficient leukocytes for study of adenine, guanine and hypoxanthine metabolism in duplicate. Suspension cultures of human lymphoblasts and leukemic cells, and monolayer cultures of skin fibroblasts and amnionic cells were studied under normal culture conditions approximately 10 cells are required for each incubation. 

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. 

Hemolysis of erythrocytes in isosmotic ammonium chloride has been used to isolate leukocytes from peripheral blood. After separation of leukocytes from blood by centrifugation through density gradients, a considerable number of contaminat-... 

Figure 5,8, CsCI profiles of compositional DNA fractions from human placenta, B mouse liver and C chicken erythrocyte, as obtained from preparative centrifugation in CS2SO4/BAMD density gradient. The rf (ligand/tiueleotide molar ratio) values used were 0.12 for mouse and 0.14 for human and chicken. Modal buoyant densities and relative amounts of the fractions arc indicated. P indicates the pellet. Notice the satellite peak (centered at abt>ut 1.700 g/ml) in the last fractions of human, and the satellite peak (1.691-1.692 g/cm ) in mouse DNA fractions i-4. A , B and C display autoradiograms of terminally labelled (Cooper et al.. 1983 1.2% agarose gels were used) Hpa 11 fragments from DNA fractions. (From Aissani...

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