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Density gradient isolation of peripheral blood mononuclear cells using a blood cell processor.
Large numbers of mononuclear cells (MNC) are needed for hematologic reconstitution using peripheral blood stem cells. The possibility of isolating those cells by discontinuous Ficoll-diatrizoate density gradient centrifugation in two blood cell processors (the Haemon tics V50 [V50] and the Cobe 2991 [2991]) were examined. Buffy coats from peripheral blood containing 6.23 X 10(8) MNC were separated in the V50, resulting in a recovery of 75 percent. The purity of the cells, defined as the percentage of lymphocytes and monocytes among all leukocytes, was 95 percent. With larger cell loads (3 to 7 X 10(9) MNC), the yield was higher in the V50 than in the Cobe 2991 (92 versus 75%). After the separation in the V50 or 2991, the cloning efficiencies of hematopoietic progenitor cells (CFU-GM and BFUe) were not different from those of cells isolated on 5 ml Ficoll-diatrizoate gradients in centrifuge tubes. Both leu apheresis and MNC separation can be carried out with the same bowl and tubing set in the V50. With that approach, an average of 6 X 10(9) MNC were processed in 16 experiments. An average recovery of 82 percent with 95 percent purity was achieved. The authors conclude that, in terms of simplicity of operation, cost-effectiveness, and maintenance of sterility, the V50 may be better suited than 2991 for the purification of MNC from peripheral blood.
PBMC ISOLATION
Whole blood contains a range of cells such as lymphocytes (B and T cells), monocytes (precursors to macrophages), granulocytes, and red blood cells. To isolate the peripheral blood mononuclear cells (PBMCs; monocytes, lymphocytes and a small percentage of other immune cells such as dendritic cells ), fresh blood is collected in EDTA (purple-capped) blood tubes or lithium/heparin (green-capped) tubes, to prevent clotting of the blood. The blood is then diluted with an equal volume of phosphate-buffered saline before layering on top of Ficoll reagent. Ficoll is a highly branched sucrose polymer that does not penetrate biological membranes.
Question.
How do you isolate peripheral blood mononuclear cells?
What are peripheral mononuclear cells?
What is the principle of PBMC isolation?
How do you separate monocytes from other cells?
What is the density gradient technique?
What is the principle of density gradient centrifugation?
What is the purpose of density gradient centrifugation in isolation of lymphocytes?
How does histopaque separate blood?
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