Hewittnoble1530

Their activation phenotype, effector function, and effector differentiation are subsequently analyzed by flow cytometry immediately or after subsequent incubation. Similarly, trogocytosis-positive cells can be identified and similarly analyzed by flow cytometry. Previous studies have described methods for analyzing T cell trogocytosis to identify antigen-specific cells or the antigenic epitopes recognized by the cells. With the current protocol, the effects of trogocytosis on the individual T cell or the ability of trog+ T cells to modulate the activation and function of other immune cells can be assessed over an extended period of time.Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such analyses. We developed a method for viral particle analysis that integrates a high-sensitivity split luciferase system and density gradient centrifugation. This protocol enables high-resolution profiling of particles produced by cells expressing Japanese encephalitis virus factors.Quantification of intestinal colonization by pathogenic or commensal bacteria constitute a critical part of the analysis to understand host-microbe interactions during different time points of their interplay. Here we detail a method to isolate non-pathogenic and pathogenic bacteria from C. elegans intestines, and classify gut phenotypes induced by bacterial pathogens using fluorescently-tagged bacteria. Furthermore, these methods can be used to isolate and identify new culturable bacterial species from natural microbiomes of wild nematodes.Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer's patch germinal center structures. Recently, we have shown that B cells responding to gut-derived antigens colonize the subepithelial dome (SED) in Peyer's patches and rapidly proliferate independently of their relative BCR affinity. To evaluate B cell proliferation within different niches in Peyer's patches, we applied in vivo EdU incorporation assay as described in this protocol.The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as pre-HSCs. Both HECs and pre-HSCs show endothelial surface phenotype and require stromal support to detect their hematopoietic activity. The method utilizing stromal cell culture followed by modified semisolid clonal culture enables us to detect the number of colony forming units for B-1/B-2 progenitors originally derived from HECs/pre-HSCs, which will reflect the potential of B-1 biased or multi-lineage repopulating HSCs.Template-based modeling, the process of predicting the tertiary structure of a protein by using homologous protein structures, is useful when good templates can be available. Indeed, modern homology detection methods can find remote homologs with high sensitivity. However, the accuracy of template-based models generated from the homology-detection-based alignments is often lower than that from ideal alignments. In this study, we propose a new method that generates pairwise sequence alignments for more accurate template-based modeling. Our method trains a machine learning model using the structural alignment of known homologs. When calculating sequence alignments, instead of a fixed substitution matrix, this method dynamically predicts a substitution score from the trained model.Kinases and ATPases perform essential biological functions in metabolism and regulation. LY3537982 order Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay. Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD+ to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.