Ficoll-Hypaque

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). 

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.

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). 

Ficoll-Hypaque Ficoll and sodium metnzoate/diatrizoate solution of density 1.077 g/mL, supplied as Ficoll Paque (Pharmacia), Histopaque (Sigma), or Lymphoprep (Nycomed AS, Pharma Diagnostic Division, Oslo, Norway), Store at 4°C protected from light. 

Prepare peripheral blood mononuclear cells (PBMC) from heparinized blood, by Ficoll-Hypaque density centrifugation (21) as follows ... 

Wash PBMC three times with 10 mL of PBS by spinning down first at 800g for 10 min, and then twice at 400g to remove excess Ficoll-Hypaque and platelets from the sample. Resuspend the cells at <107/mL in PBS/BSA. 

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. 

Ficoll-Hypaque preparation (Accurate Chemical and Scientific Corporation, Westbury, NY). 

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. 

Warm required amount of ficoll-hypaque for 15 min. Then add 15 mL to each 50 mL conical centrifuge tube. 

Withdraw blood from the heparinized patient blood sample (see Note 1) and gently place onto the top of the ficoll-hypaque. Spin the tubes at 500g for 25 min at 25°C (room temperature). 

Remove the cell layer with as little ficoll-hypaque as possible. 

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). 

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. 

Fig. 1. Recovery of MCF-7 cells seeded at a concentration of 0.13% in buffy coat samples as a function of hematocrit. Samples were loaded on a Ficoll-Hypaque gradient at the concentration specified by the experimental point.

Ficoll-Hypaque to isolate lymphocytes Digest lymphocytes in detergenl/proteinase K... 

The reactivity of either blood or bronchoal-veolar lymphocytes to beryllium can be determined using the LPT. To test peripheral blood, a sample of heparinized blood should be centrifuged over Ficoll-Hypaque to obtain blood mononuclear cells (-70% lymphocytes and 30% monocytes). Cells obtained by bronchoalveolar lavage can be used after washing (Rossman et al. 1988). While the major published studies have utilized bronchoalveolar cells in this manner, occasionally other techniques are employed to enhance the proliferative response of... 

PMN and mononuclear leukocytes (MNL) were prepared from Ficoll-Hypaque density difference centrifugation as previously described by English (1976). After isolation, the cells were enumerated and diluted to desired concentrations in Hank s balanced salt solution (BBSS), Grand Island Biological Company, Grand Island, New York. 

In order to systematically investigate the mechanism(s) of fragile chromosome expression in folate deficient media we developed a basic culture medium. The medium is deficient in folic acid and a number of metabolites produced by folate mediated one-carbon transfer reactions (Fig. 1). These include the amino acids, serine, glycine and methionine the purine source hypoxanthine and the thymidylate source thymidine. Previous studies of fragile site expression have used leucocytes isolated in autologous plasma and cultured in medium supplemented with undialyzed serum. In order to eliminate serum sources of folate, amino acids and nucleotide precursors, we have used washed lymphocytes isolated from ficoll-hypaque banding and medium containing dialyzed human AB serum. 

Measure 150 ml Ficoll-Hypaque into a 600 ml transfer pack and allow to run into processing bag (P) through one line. Use clamps throughout the procedure to direct the flow of liquids to the appropriate bags. 

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