Ficoll-Paque

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.

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 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. 

A more direct method recommended by Pharmacia (Appendix 3) uses 2 ml whole anticoagulant-treated blood diluted with an equal volume of BSS. This is layered over 3 ml Ficoll-Paque and sedimented at 400 g for 30 min. The lymphocytes may be removed from the interphase and the upper layer may be used as a source of autologous plasma. 

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... 

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

You will need mononuclear cells from peripheral blood or bone marrow collected in heparinized glass tubes and isolated on Ficoll-Paque as rapidly as possible. After collecting the interphase cells, they are washed and resuspended in the buffered medium supplemented with 10% FCS (= assay medium) and kept on melting ice until analysis. Alternatively, the cells are frozen in medium with 10% DSMO and at least 10% FCS in liquid nitrogen according to protocols for cryopreservation (see Note 1). 

Carefully layer the whole blood onto an equal volume of Ficoll-Paque (see Note 5). 

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. 

On d 0 leave Ficoll-Paque to warm to room temperature if stored at +4°C. 

Invert the Ficoll-Paque and check that it has reached room temperature and add 15 mL to four sterile centrifuge tubes. If the buffy coat has a large volume (>80 mL), then steps 5-13 can be repeated if the maximum number of PBMCs present are required. 

Using a 10 mL plastic pipet, carefully layer 25 mL of the buffy coat-RPMI mixture on top of the Ficoll-Paque. This is achieved by allowing the buffy coat to run slowly down the inside of the centrifuge tube. The centrifuge tube should contain no more than 40 mL at this stage. 

Isolate PBMC using the standard method of density gradient centrifugation on Ficoll-Paque PLUS, following the vendor s recommendations (http //www.stemcell.com/ /media/ Technical%20Resources/l/F/F/B/3/29630 PIS 2 l 2. ashx). 

During the dextran sedimentation, prepare the following in 50-mL conical tubes (1) 35 mL of 0.09% NaCl, (2) 20 mL of 1.7% NaCl, (3) 12 mL of Ficoll-Paque , and (4) 20 mL of injection- or irrigation-grade water. These solutions are normally stored at 4 °C, but to prevent priming and aggregation of PMNs, it is optimal to warm the solutions to room temperature. 

Underlay 10 mL of Ficoll-Paque beneath the NaCl cell suspension. Spin at 500-700 X g (with low or no break) for 25 min at room temperature. 

---