Ficoll gradient

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. 

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. 

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). 

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). 

Isolation of human neutrophils. Leukocytes were obtained from normal donors by leukapheresis with an IBM 2997 Blood Cell Separator (8). Normal mature neutrophils were purified from this mixed leukocyte preparation by dextran sedimentation and Ficoll-Hypaque gradient centrifugation as previously described (9). The Wright-stained smears of the preparation showed that the cells were over 95% neutrophils. 

In mammals, red blood cells do not contain DNA since they are devoid of nucleus. The hemoglobin in them can get adsorbed to DNA if it is present during the isolation procedure. Hence for the isolation of DNA from blood, red blood cells are first removed either by Ficoll/Hypaque gradient centrifugation, or lysed by the detergent Triton X-100 followed by recovery of nuclei of white blood cells, which carry DNA. 

In order to stabilise the sedimenting cells, it is necessary to include a gradient and those most commonly used are serum or Ficoll (Pharmacia Ltd.) (Boone et al., 1968 Miller and Phillips, 1969 Macdonald and Miller, 1970 Warmsley and Pasternak, 1970). Sucrose has also been used (Sinclair and Bishop, 1965 Morris et al., 1967 Shall and McClelland, 1971 Shall, 1973b) but tends to lower cell viability, unless great care is taken to maintain isotonicity. 

Boone et al. (1968) centrifuge cells through a discontinuous 10-20% Ficoll gradient made up in Eagle s minimum essential medium modified for suspension (i.e. lacking calcium and bicarbonate and containing 10 times the normal phosphate concentration). They use an A-1X zonal centrifuge rotor and spin for 1 h at 1000 r.p.m. at 20°C, and obtained clear separation of different cell types (HeLa and rabbit thymocytes). 

Pretlow et al. (1978) showed that a shallow gradient of Ficoll (2.7-5.5%) centrifuged at low speed (about lOOOg) for about 100 min, allowed clear, isokinetic separation of spheres differing 2-fold in volume. The density change in the gradient was small (1.017-1.027 g/ml) and viscosity effects negligible. 

The LACS cell separator is marketed by Medilog (Appendix 3). This simply consists of a separating chamber on which a 360 ml Ficoll gradient (2.5-7.5%) is made and a cell suspension is layered onto the gradient through a sieve. 

The method contains three stages including first establishing a cell line followed by test chemical exposure and finally evaluated for expression of cell surface markers. To establish a cell line, human leukocyte preparations are attained from a plasma distributor. The leukocyte preparations as described by Ryan et al. (2004), are diluted with an equal part of complete medium (RPMI 1640 containing 1 x L-glutamine, 1 x penicillin-streptomycin-neomycin antibiotic mixture), 30 p,2-mercaptoethanol and 10 % heat inactivated fetal bovine serum. The diluted preparation is layered onto a Ficoll-Paque gradient to... 

Figure 2. Electron micrograph of a synaptosome fraction isolated from mouse brain by Ficoll density-gradient centrifugation. SV, synaptic vesicles M, mitochondria SJ, synaptic junction with attached PSyD. Bar = 0.5 mm. (From Schrimpf et at 2005)...

Fig. 1. Electron micrograph of synaptosomes isolated from rat brain by differential and Ficoll-gradient centrifugation (Courtesy of Christiane Walch-Solimena)...

Synaptosomes are enriched in the 9 % Ficoll layer, or more specifically, at the interfaces between the 12 % and 9 %, and the 9 % and 6 % Ficoll layers. Myelin is enriched at the top of the gradient whereas most of the mitochondria are in the pellet. Collect the fraction enriched in synaptosomes and dilute to a final volume of 10 ml in assay buffer (solution 4). At this point, the protein concentration should be determined, e.g., by the method of Bradford (1976). 

Separation of Lymphocytes from Human Umbilical Cord Blood Using Ficoll Hypaque Gradients... 

Carefully layer the diluted blood onto Ficoll-Hypaque gradients (1 3 ratio) in a 15-mL conical culture tube (3 mL blood onto 9 mL Ficoll-Hypaque). 

The most common materials used to generate density gradients are sucrose, Ficoll, and Cs salts. Sucrose solutions can be as concentrated as 65% w/w, with a maximum density of 1.32 g/cm3 at 4 °C however, concentrated solutions of sucrose have high osmotic strength and viscosity. Ficoll is a brand name for a synthetic polysaccharide with an average MW of 400,000 and a maximum density in aqueous solution of 1.23. It is very useful to separate osmotically sensitive particles, like mammalian cells. 

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. 

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