KOSEN 한인과학기술자네트워크

분리가 쉽지는 않습니다..Nycomed사의 chemotaxis assays-Polymorphprep kit를 이용하시는 편이 가장 손쉬울 것 같네요.. 아래는 많아 사용하는 percoll법입니다. Neutrophil Isolation Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation of erythrocytes and density-gradient centrifugation of leukocytes. All procedures up to and including density-gradient centrifugation were conducted at 22° C unless stated otherwise. Blood was anticoagulated with acid-citrate-dextrose and centrifuged at 400 × g for 20 min to remove platelets. The platelet-rich plasma supernatant was removed and centrifuged at 2,000 × g for 30 min to prepare platelet-poor plasma (PPP). The platelet-depleted cell precipitate from the first centrifugation step was mixed with phosphate-buffered saline (PBS) (1:4). Dextran (Baxter, Warrington, UK) was added to a final concentration of 2% and erythrocytes were sedimented under gravity for 45 min at 15° C. The erythrocyte-depleted supernatant containing leukocytes was centrifuged at 400 × g for 10 min. The resulting leukocyte-enriched pellet was resuspended in PPP. The leukocytes were then layered over a discontinuous gradient of Percoll (4 ml of 61.5% and 4 ml of 76%) in a 15-ml polypropylene centrifugation tube. The Percoll densities were created from pure Percoll diluted with 10× PBS and PPP. Final percentages of PPP (vol/vol) in the Percoll solutions were 42.89% and 23.97% for the 61.5% Percoll layer and 76.0% layer, respectively. The Percoll densities and layered cells were then centrifuged at 400 × g for 20 min. After centrifugation, mononuclear cells were found at the plasma-61.5% Percoll layer interface, and neutrophils were found at the 61.5-76.0% Percoll layer interface. Neutrophils were removed and washed twice in RPMI-1640. Cells were counted with a hemocytometer and resuspended at 106 cells/ml. Cells were plated with the compounds investigated in the study in RPMI-1640 containing 10% autologous-plasma-derived serum (PDS). PDS was obtained from nonanticoagulated cell-free plasma. Cell-free plasma was obtained by centrifugation of whole blood immediately after venipuncture. The cell-free plasma was incubated at 37C and PDS was derived by removal of the protein clot. The purity of neutrophils was consistently greater than 99%. >neutrophil의 분리 방법과 (rat의 blood나 복강에서...) >어느정도로 순수하게 neutrophil로 분리되었는지 확인할수 있는 방법 부탁드립니다...