Notes

Statement of intra- as well as intermolecular vibrational coherences from the aqueous tryptophan significant activated by simply photodetachment.
Here, we describe a detailed protocol for the isolation of purified populations of viable spermatogenic cells derived from the non-human primate model organism Macaca fascicularis (cynomolgus). ACY-1215 order Using fluorescence-activated cell sorting (FACS), we describe methods to isolate spermatogonia and primary spermatocytes ranging across the sub-stages of meiosis prophase I. These cell populations can be used with a variety of downstream assays, including single-cell approaches such as RNA sequencing, chromatin immunoprecipitation, quantitative RT-PCR, and immunocytochemistry. For complete details on the use and execution of this protocol, please refer to Lau et al. (2020).Drosophila larval musculature is a genetically and optically accessible system to study muscle development. Each larval muscle is a single fiber with conserved cytoarchitecture, including its sarcomere structure and composition. Here, we present a workflow for systematically analyzing muscle structure and function at discrete larval stages, as well as throughout the larval instars, using both newly developed and adapted methods. For complete details on the use and execution of this protocol, please refer to Balakrishnan et al. (2020).Effective therapeutics for malignant primary brain tumors, such as glioblastomas (GBMs), are urgently needed. To facilitate and expedite early-phase GBM therapeutic development, we describe a protocol that allows the intranasal delivery of experimental compounds in GBM orthotopic mouse models. Compounds delivered through this route can bypass the blood-brain barrier and thus help validate effective therapeutic targets for GBMs. For complete details on the use and execution of this protocol, please refer to Pinkham et al. (2019).Open or accessible regions of the genome are the primary positions of binding sites for transcription factors and chromatin regulators. Transposase-accessible chromatin sequencing (ATAC-seq) can probe chromatin accessibility in the intact nucleus. Here, we describe a protocol to generate ATAC-seq libraries from fresh Arabidopsis thaliana tissues and establish an easy-to-use bioinformatic analysis pipeline. Our method could be applied to other plants and other tissues and allows for the reliable detection of changes in chromatin accessibility throughout plant growth and development. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).The discovery of potent cell-permeable E3 ubiquitin ligase ligands can significantly facilitate the development of proteolysis targeting chimeras (PROTACs). Here, we present a protocol to determine the binding affinity of ligands toward CRBN E3 ubiquitin ligase, using a cellular target engagement mechanism and in-cell ELISA assay. This protocol is easy to establish, with relatively low cost and rapid time frame. It can also be modified to measure the level of other proteins or determine the ligand affinity toward other E3s. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).This protocol describes a standardized method for analyzing Drosophila behavioral rhythmicity under light dark cycles, temperature ramps, and free running conditions. The protocol constitutes a step-by-step guide from generation of appropriate Drosophila genetic crosses to behavioral experiments. We also provide an open-source computational framework using R for the analysis of the phase of behavior using circular statistics. An extended method for complete use is also provided. For complete details on the use and execution of this protocol, please refer to Fernandez et al. (2020).This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.The elastic properties of cell membranes, particularly the membrane tension and bending modulus, are known to be key regulators of cellular functions. Here, we present a correlative and integrated tool based on optical tweezers and scanning electron microscopy to accurately determine these properties in a variety of cell types. Although there are intrinsic difficulties associated with correlative experiments, we believe that the methods presented can be considered a suitable protocol for determining the elastic properties of cell membranes. For complete details on the use and execution of this protocol, please refer to Soares et al. (2020).Cytosolic Ca2+ levels are maintained at low nanomolar concentrations, and disruption of Ca2+ homeostasis is associated with cell/tissue damage. Thus, methods have been developed to accurately assess cellular Ca2+ levels, each with intrinsic advantages and disadvantages. Here, we present in detail a ratiometric fluorometric method for cytosolic Ca2+ measurement in cultured melanoma cells using Fura 2-AM cell loading and fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Esteves et al. (2020).The classical aortic ring model is well suited for deciphering pro-angiogenic processes. Here, we propose simple modifications of the standard protocol to study various anti-angiogenic processes from growth arrest to capillary degeneration. Aortic rings are cultured under basal conditions for 6 days to allow physiological vessel sprouting and then split into treatment groups to follow capillary growth or degeneration for an additional 2 days.Targeted identification of cellular processes responsible for a phenotype is of major importance in guiding efforts in bioengineering and medicine. Genome-scale metabolic models (GEMs) are widely used to integrate various types of omics data and study the cellular physiology under different conditions. Here, we present PhenoMapping, a protocol that uses GEMs, omics, and phenotypic data to map cellular processes and observed phenotypes. PhenoMapping also classifies genes as conditionally and unconditionally essential and guides a comprehensive curation of GEMs. For complete details on the use and execution of this protocol, please refer to Stanway et al. (2019) and Krishnan et al. (2020).
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