During swarming, high density flagella-driven bacteria migrate collectively in a swirling pattern on wet agar surfaces, immersed in a thin viscous fluid layer called "swarm fluid". Though the fluid environment has essential role in the emergence of swarming behavior, the microscopic mechanisms of it in mediating the cooperation of bacteria populations are not fully understood. Here, instead of micro-sized tracers used in previous research, we use gold nanorods as single particle tracers to probe the dynamics of the swarm fluid. This protocol includes five major parts (1) the culture of swarming bacterial colony; (2) the preparations of gold nanorod tracers and the micro-spraying technique which are used to put the nanotracers into the upper fluid of bacterial swarms; (3) imaging and tracking; (4) other necessary control experiments; (5) data analysis and fitting of physical models. With this method, the nano-sized tracers could move long distances above motile cells without direct collisions with the bacteria bodies. In this way, the microscopic dynamics of the swarm fluid could be tracked with high spatiotemporal resolution. Moreover, the comprehensive analysis of multi-particle trajectories provides systematic visualization of the fluid dynamics. The method is promising to probe the fluid dynamics of other natural or artificial active matter systems.Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA's activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA's modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA's substrate Rab33b. Prior to the identification and publication of SidJ's activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA's activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.Giant unilamellar vesicles (GUVs) are a widely used model system for a range of applications including membrane biophysics, drug delivery, and the study of actin dynamics. While several protocols have been developed for their generation in recent years, the use of these techniques involving charged lipid types and buffers of physiological ionic strength has not been widely adopted. This protocol describes the generation of large numbers of free-floating GUVs, even for charged lipid types and buffers of higher ionic strength, using a simple approach involving soft polyacrylamide (PAA) gels. This method entails glass cover slip functionalization with (3-Aminopropyl)trimethoxysilane (APTES) and glutaraldehyde to allow for covalent bonding of PAA onto the glass surface. After polymerization of the PAA, the gels are dried in vacuo. https://www.selleckchem.com/products/pf-05251749.html Subsequently, a lipid of choice is evenly dispersed on the dried gel surface, and buffers of varying ionic strength can be used to rehydrate the gels and form GUVs. This protocol is robust for the production of large numbers of free-floating GUVs composed of different lipid compositions under physiological conditions. It can conveniently be performed with commonly utilized laboratory reagents.Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope.