Ficoll

M. P. Bohrer, G. D. Paterson, P. J. Carrol. Hindered diffusion of dextran and ficoll in microporous membranes. Macromolecules 77 1170-1173, 1984. 

H. A. Beequerel (ficole Polytechnique, Paris) discovery of spontaneous radioactivity. 

G. Charpak (ficole Superieure de Physique et Chemie, Paris, and CERN Geneva) invention and development of particle detectors, in particular the multiwire proportional chamber. 

Mitchell, G.F. and Lewers, H.M. (1976) Studies on immune responses to parasite antigens in mice IV. Inhibition of an anti-DNP antibody response with antigen, DNP-Ficoll containing phosphorylcholine. International Archives of Allergy and Applied Immunology 52, 235—240. 

Figure 9.5 Healthy volunteer monocytes after erythrophagocytosis. Rabbit erythrocytes were incubated with heat-inactivated mouse antirabbit erythrocyte serum. Human peripheral blood monocytes (MN) were obtained after Ficoll-Paque isolation and monocyte clumping with subsequent separation from lymphocytes, yielding a 95 % pure MN population. Non-ingested erythrocytes were removed by hypotonic lysis.

Heparinized blood samples may be stored at 4°C for up to 48 h without affecting the SCE response (Lambert et al., 1982). If the test agent is known to react with serum or red blood cells, the mononuclear lymphocytes may be isolated by use of a Ficoll/Hypaque gradient (Boyum, 1968). 

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. 

Figure 7.7. Expression of CR3 in neutrophils in unfractionated blood and after isolation. The expression of CD1 lb was measured in neutrophils in (i) unfractionated whole blood, (ii) after isolation using Mono-Poly resolving medium and (iii) after isolation using dextran sedimentation and centrifugation in Ficoll-Hypaque. Further experimental details may be found in Watson, Robinson Edwards, 1992.

Take 20ml of ficol (Pharmacia) in a 50ml Falcon tube (5 tubes). 

Fig. D.l Ficol gradient for enrichment of peripheral blood lymphocytes.

Ficoll Paque density gradient (GE Healthcare, Piscataway, NJ). 

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. 

Ficoll-hypaque lymphocyte separation medium (Organon Teknika Corp., Durham, NC). 

Add ficoll-hypaque lymphocyte separation medium to tubes, and carefully layer the plasma fraction over the ficoll-hypaque medium at a final volume ratio of 2 parts ficoll-hypaque 3 parts plasma. If 50-mL conical polypropylene centrifuge tubes are used, add 20 mL of ficoll hypaque to the tube first then layer 30 mL of plasma on top of the ficoll hypaque such that a sharp interface is visible between the two layers. 

After centrifugation, carefully pipet away the platelet- and mononuclear cell-containing layer, which forms at the plasma-ficoll-hypaque interface. Pipet away the remainder of the supernatant, and resuspend the pellet in 2-3 mL of PBS. 

Ficoll-hypaque, Lymphocyte Separation Medium (Organon Teknika, Dnrham, NC). 

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). 

Developed method of two-step centrifugation of cells enables the isolating up to 95% of CD 45 - cells and up to 97% of CD 34 -cells from cord blood (Fig. 1). It is worth of noting that ficoll causes the losses in the number of isolated cells (up to 55%). Therefore ficoll use to isolate cells is not absolutely positive. 

It should be emphasized that number of CD 34 - cells is significantly higher of their absolute content in cell concentrate, obtained using ficoll and frozen with PEO-1500 under analogous condition, that also testifies to a considerably higher quality of NCs concentrate our method. 

Microspheres and nanoparticles often consist of biocompatible polymers and belong either to the soluble or the particle type carriers. Besides the aforementioned HPMA polymeric backbone, carriers have also been prepared using dextrans, ficoll, sepharose or poly-L-lysine as the main carrier body. More recently alginate nanoparticles have been described for the targeting of antisense oligonucleotides [28]. As with other polymeric carrier systems, the backbone can be modified with e.g. sugar molecules or antibody fragments to introduce cellular specificity. 

Primary CLL cells purified from peripheral blood of volunteer patients (see Note 3) CLL cells are purified by Ficoll gradient centrifugation, by negative selection, or by a combination of both (see Note 4). CLL cells can be used immediately or frozen in 90% heat/inactivated FBS supplemented with 10% DMSO and stored at -80°C or in liquid nitrogen for short-and long-term storage, respectively. CLL cells are cultured in complete RPMI medium and maintained in a humidified atmosphere of 5% CO at 37°C (see Note 5). 

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