Centrifugation gradient, Sedimentation

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. 

Diffusion is driven by a concentration gradient sedimentation is driven by a centrifugal field. Why should the transport coefficient ratio s/D depend only on nontransport quantities ... 

Fig. 15. Polysomal mRNP from reticulocytes. A. Sucrose gradient sedimentation of the polyribosomal suspension after EDTA treatment (40 hours centrifugation). B.Sucrose gradient sedimentation of unlabeled, sodium dodecyl sulfate-extracted polyribosomal RNA supplemented with the high specific radioactivity RNA detached from labeled polyribosomes by EDTA treatment. C. CsCi equilibrium sedimentation of the messenger ribonucleoprotein complex. Ribosomal subparticles have been added as Internal markers. The centrifugation was carried out at 4°C for 18 hours at 36,000 rpm in an SW-39 rotor. (From Huez et al. 1967. Biochim. Biophys. Acta, 145 629-636 Burny et al. 1969. Biochim, Biophys. Acta, 190 228-231.)...

Fig. 5. DNA strand breaks produced by a 30 min exposure of A549 cells to BLM (500 Mg ml ). Strand breaks were assayed by alkaline sucrose gradient centrifugation [14]. Sedimentation was from right to left. Immediately after BLM treatment O 2 h recovery, 0 mM 3AAB 2 h recovery, 5 mM 3AAB...

In isopycnic centrifugation, particles sediment in a density gradient until such time as they reach a region of density equal to their own density, as can be seen from equation (1), the sedimentation velocity becomes zero under these conditions. The experimental procedure is summarized in Figures 4 and S. 

If a sedimentation experiment is carried out long enough, a state of equilibrium is eventually reached between sedimentation and diffusion. Under these conditions material will pass through a cross section perpendicular to the radius in both directions at equal rates downward owing to the centrifugal field, and upward owing to the concentration gradient. It is easy to write expressions for the two fluxes which describe this situation ... 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

If the concentration profile can be determined the moduli can be evaluated. In principle there is no reason why this should be a non-linear measurement, it depends upon the magnitude of the gravitational Peclet number. Buscall35 suggested that a low speed centrifuge could be used to apply different acceleration gradients to the dispersion. If the angular velocity of the rotor is cor and if X is the distance from the centre of the rotor to the top of the sediment then the pressure balance equation becomes... 

Regardless of the rotor speed and maximum velocity, sedimentation (or flotation) will not occur in a solution of equal density to the sample. Iso-density methods use this lack of movement in a manner comparable to a pH gradient in iso-electric focusing techniques. The methods are a combination of sedimentation and flotation, achieved by using a density gradient that straddles the density of the particles concerned. On centrifugation, the particles sediment until they reach a solvent zone with the same density. This results in the development of a zone for each type of particle present in the sample. 

Another technique consists of submitting the emulsion to centrifugation and determining the droplet volume fraction < / at the top (bottom) of the cream (sediment). The centrifugation typically takes several hours until the equilibrium volume fraction is achieved. After equilibration, if the droplets occupy a distance much less than that of the centrifuge lever arm, the spatial gradient in the acceleration can be neglected, and the osmotic pressure can be determined (see Fig. 4.1) ... 

Equilibrium sedimentation technique working with a multi-component solvent forming a density gradient in a centrifugal field... 

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