Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood....Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood. We here develop a simple and quick method for the purification of viable leukocytes from whole blood using novel tools, named tLeukoCatch (tip-type) or sLeukoCatch (syringe-type), which is equipped with three Pall filter layers and captures leukocytes but not red blood cells (RBCs) in whole blood. Indeed, we showed that several million leukocytes per mL (~35% of the recovery rate) were captured and eluted from whole blood. The number of contaminant RBCs decreased from several million to several thousand. When mouse blood was hemolysed, almost all of the lysed RBC fragments were removed by passage through sLeukoCatch. Optical microscopic observation confirmed that the recovered leukocytes were sufficiently healthy to respond to growth stimuli. Efficient leukocyte recovery was also confirmed for hemolysed human blood. These results suggest that the LeukoCatchTM system is useful for bedside diagnosis and basic research with blood samples.展开更多
Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunopreci...Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.展开更多
文摘Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood. We here develop a simple and quick method for the purification of viable leukocytes from whole blood using novel tools, named tLeukoCatch (tip-type) or sLeukoCatch (syringe-type), which is equipped with three Pall filter layers and captures leukocytes but not red blood cells (RBCs) in whole blood. Indeed, we showed that several million leukocytes per mL (~35% of the recovery rate) were captured and eluted from whole blood. The number of contaminant RBCs decreased from several million to several thousand. When mouse blood was hemolysed, almost all of the lysed RBC fragments were removed by passage through sLeukoCatch. Optical microscopic observation confirmed that the recovered leukocytes were sufficiently healthy to respond to growth stimuli. Efficient leukocyte recovery was also confirmed for hemolysed human blood. These results suggest that the LeukoCatchTM system is useful for bedside diagnosis and basic research with blood samples.
文摘Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
