Mononuclear cell fraction centrifugation

Enrichment of rare cancer cells from peripheral blood samples is an application that typically requires density gradient centrifugation as a first step. Application of this technique addresses two objectives depletion of erythrocytes and depletion of polymorphonuclear cells. It is expected that cancer cells undergo sedimentation with the mononuclear cell fraction because of their similar density. However, some studies have found that cancer cells are also lost in the polymorphonuclear fraction or the erythrocyte fraction (8,9). Optimization of the density gradient sedimentation step is an important issue in such an application, because it will determine the recovery of rare cells from blood and affect the chances of their detection by immunochemical means. 

Transfer the mononuclear cell fraction to a sterile 50-mL screw-cap centrifuge tube containing 35 mL of PBS P/S. 

Peripheral mononuclear cells (PBMC) were isolated from heparinized blood by standard gradient centrifugation (B yum, 1968). T-enriched cell fractions were obtained by resetting PBMC with 2-aminoethylisotbiouronium bromide-treated sheep red blood cells (Madsen and Jobnsen, 1979). Cell suspensions were adjusted to a concentration of 5 x 10 cells/ml and sonicated at 23 kc/sec. 

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