Cell separation ficoll

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. 

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. 

Lymphoprep (Ficoll-Paque Pharmacia Biotech, Uppsala, Sweden) or similar cell-separation medium. Storage according to manufacturer s recommendations. 

Vacutainer CPT cell preparation tubes (Becton Dickinson) can be used as an alternative to the standard Ficoll-Paque protocol for lymphocyte isolation. This allows for sterile blood collection and cell separation using a single centrifugation step. 

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. 

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). 

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). 

Separate rosetted cells by layering the cell sample on a 24% Ficoll soludon centrifuge the mixed cell preparadon (lOOOg) for 30 min at 4 C. Remove nonrosetdng cells from the interface and wash by centrifugation in PBS. 

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. 

A major consideration in transcriptomics studies is the source of the RNAused. Peripheral blood mononucleocytes (PBMCs) are widely used, but the Ficoll separation to remove neutrophils and red blood cells also introduces errors related to cellular response to this manipulation and time ex vivo [9]. An advantage of PBMCs is that they can be cry op reserved for latter examinations (see Note 1). PAXgene blood tubes collect the blood directly from the patient into fixative, are widely used in clinical settings, and provide reproducible results [10]. 

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. 

Cultures of PHA blasts are initiated by purifying peripheral blood mononuclear cells, usually by Hypaque-Ficoll density gradient centrifugation. These cells are then incubated with 10 pg/mL of the plant lectin phytohemagglutinin (PHA), which activates T cells. After 48 h, the medium is removed and fresh medium, supplemented with IL-2, is added. The cells are replenished with fresh medium and IL-2 every 3 d. These cells may be infected with either cell-free virus or with HIV-infected cells. Peak infection usually occurs anywhere from 7-28 d postinfection, depending on the size of the inoculum and the characteristics of the virus. In general, at the peak of infection, <5% of the cells in the culture are actually infected. However if extreme care is taken and the T-cell blasts are separated from nonactivated cells, it is possible to obtain infection in the majority of the cells in the culture (58). 

Figure 14. Electrophoretic separation of rabbit and human erythrocytes. A mixture of 4 x 107 rabbit and 5 x 107 human erythrocytes suspended in 5 ml 1.5% Ficoll 70 are layered to a linear 3-13% Ficoll gradient of 75 ml followed by an overlay of 5 ml, and electrophoresed at 4°C and 250 mA for 10 min. Fractions of 1 ml each were taken. The contribution of the two cell types was estimated by electronic sizing. The black bar represents initial cell band-width. (O——O) Rabbit erythrocytes ( - ) human erythrocytes.

Figure 15. Electrophoresis of murine spleen cells. Murine (CBA strain) spleen cells (4 x 107) suspended in 5 ml 1.5% Ficoll are layered on a 3-13% Ficoll density gradient followed by an overlay of 5 ml. Electrophoresis proceeds for 15 min at 250 mA and 4°C. Fractions of 1-2 ml are taken. For comparison, in the inset the separation of CBA mouse spleen cells by free-flow electrophoresis is given. [Redrawn from Zeiller and Pascher (1973) with kind permission of Dr. K. Hannig and Verlag Chemie.]...

Figure 16. Electrophoretic separation of murine (GR strain) lymphoid cells. Unless otherwise stated, cells suspended in 5 ml 1.5% Ficoll were electrophoresed for 25 min at 250 mA at...

Mononuclear cells from IM patientsy patients with myeloid proliferation and controls were obtained by Ficoll-Isopaque gradient separation (5) of heparinized peripheral blood. 

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