Isolation techniques of neutrophils and peripheral blood mononuclear cells for the comparative experiments in humans and pigs model organisms in flow cytometry

The pig is due to the morfology of their bodies often used as a model organism from the experimental organisms for comparison with man. The aim of this study ist o determine which isolation technique can be used for comparative imunological experiments focused on human and porcine neutrophils and monocytes . In comparative studies, it is necessary that both cells compared species were isolated by the same technique. Selected isolation techniques are the first step for subsequent detection of selected parameters in flow cytometry and for cultivation of peripheral blood mononuclear cells to profit monocyte-derivated macrophages. For isolation of neutrophils were chosen these techniques: isolation of double density gradient centrifugation Histopaque 11191 a 1077, isolation of double density gradient centrifugation Histopaque 11191 a 1077 after dextran and only dextran sedimentation. Monocytes were isolated by density gradient centrifugation Histopaque 1077, density gradient centrifugation Histopaque 1077 with immunomagnetic separation of CD14+ cells, double density gradient centrifugation Histopaque 11191 a 1077 and double density gradient centrifugation Histopaque 11191 a 1077 after dextran sedimentation. For the isolation of neutrophils is the best tested technique dextran sedimentation. For the isolation of monocytes from peripheral blood show the best results density gradient centrifugation Histopaque 1077. Techniques were evaluated by purity and yield isolated cells during comparison both organisms. Key-Words: dextran sedimentation, density gradient isolation techniques, clearance, yield, CD14+ Introduction Flow cytometry is a highly sophisticated method capable of sorting cells according to their size and granularity and subsequently selected by marking of selected cells surface receptors. Flow cytometry are often analyzed blood samples. Therefore, the work addresses the isolation of neutrophils and monocytes from peripheral blood. Due to the fact that the pig is often used as a model organism for comparative studies of human medicine is this work aimed at two organisms human and pig. The aim was to choose the technique that will be most suitable for isolating cells of the immune system needs further study in the analysis of flow cytometry. As a cells of interest were chosen human and porcine neutrophils and monocytes. This thesis is focused on the comparison of isolation techniques in terms of purity and yield of the isolated cells. Whereas in comparative studies must follow the same procedure for isolating cells from both species were the most successful methods chosen which showed high purity and yield in both species. Material and Methods For isolation of neutrophils and monocytes in pigs has been used 10 healthy pigs stabled in the experimental barn in Veterinary Research Institute in Brno. Pigs were fed a standard diet. Peripheral blood was collected them in the morning, from vena cava cranialis. Human neutrophils and monocytes were isolated from peripheral blood of 10 healthy individuals. Cells were isolated from heparinanticoagulated blood. For isolation of neutrophils were chosen these techniques: isolation of double density gradient centrifugation Histopaque 11191 a 1077 (sterile-