Ficoll-Hypaque gradient centrifugation

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. 

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. 

Small scale preparation of mononuclear cells (MNC) by Ficoll-Hypaque gradient centrifugation... 

Small scale preparation of mononuclear cells (MNC) by Ficoll-Hypaque gradient centrifugation 385 Preparation of tymphocytes for analysis of intracellular cytokines in vitro 386 Staining for intracellular qrtokines in activated mononuclear cells in vitro 387 Preparation of monocytes for measiuement of intracellular cytokines in vitro 388... 

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). 

Mixed leukocytes are prepared using dextran sedimentation of erythrocytes, centrifugation of leukocytes from the plasma, and lysis of residual erythrocytes with ammonium chloride. Granulocytes and mononuclear leukocytes are separated from each other and from erythrocytes by density gradient centrifugation using a mixture of Ficoll, Hypaque and dextran. Thirty ml of blood provides sufficient leukocytes for study of adenine, guanine and hypoxanthine metabolism in duplicate. Suspension cultures of human lymphoblasts and leukemic cells, and monolayer cultures of skin fibroblasts and amnionic cells were studied under normal culture conditions approximately 10 cells are required for each incubation. 

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