Author/Editor		Hajdinjak, Tine
Title		Priprava dendritskih celic iz periferne krvi
Type		monografija
Place		Ljubljana
Publisher		Univerza v Ljubljani, Medicinska fakulteta
Publication year		2000
Volume		str. 63
Language		slo
Abstract		Introduction In cancer immunotherapy and other fields of immune system modulation, dendritic cells (DC) are gaining more and more importance. They bridge innate and specific immunity by influencing NK cells. They are the only antigen-presenting cells capable of initiating a new T cell response, but they can also induce tolerance. Purpose We adopted and modified different methods for DC production. We studied the possibility of correlating the actual form of DC to their phenotype. DC development in vivo is subdivided in many stages, while in vitro only immature and mature forms are acknowledged. We supposed that it shoud also be possible to identify different immature stages of DC in vitro. In one-way allogeneic MLR, a basic functional test, the stimulating capacity of DCs is typically evaluated on day 5 or 6 of the cell culture. We anticipated that due to the stimulating properties of DCs, it would be possible to reveal significant proliferative responses earlier than that, thus reducing the performing time for the test. Materials and methods We prepared DCs in cell cultures using mononuclear cells, isolated from buffy coats from healthy blood donors. Monocytes were enriched with the use of Dynal immunomagnetic beads. We used calcium ionophores and cytokines IL-4, GM- CSF and TNFalpha. Using light microscopy and phase-contrast, morphological features of monocyte to DC transformation were followed. The expression of leukocyte antigens and the ability of phagocytosis were assessed with the use of flow cytometry. Transmission electron microscopy and classic immunofluorescence microphotographs of DCs were also taken. In one-way allogeneic MLR, the kinetics of proliferative response was followed with the the use of a liquid (beta-scintillation counter to measure the DNA incorporation of 3H- thymidine. Results The cell culture medium with cytokines should contain IL-4 at 800U/ml and GM-CSF at 800U/ml in a 6-day culture. (Abstract truncated at 2000 characters).
Descriptors		DENDRITIC CELLS CELLS, CULTURED INTERLEUKIN-4 LEUKOCYTES, MONONUCLEAR GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR TUMOR NECROSIS FACTOR BLOOD DONORS IONOPHORES CALCIUM FLOW CYTOMETRY MICROSCOPY, ELECTRON MICROSCOPY, FLUORESCENCE