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This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes. For complete details on the use and execution of this protocol, please refer to Meul et al. (2020).This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment. For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).The lack of advanced in vitro models recapitulating the human brain complexity is still a major obstacle in brain development and neurological disease research. Here, we describe a robust protocol to derive human midbrain organoids from neuroepithelial stem cells. These complex 3D models are characterized by the presence of functional neurons, including dopaminergic neurons and glial cells, making them particularly attractive for the study of Parkinson disease. For complete details on the use and execution of this protocol, please refer to Monzel et al. (2017).This protocol entails a simple method for isolation, culturing, and in vitro differentiation of adult neural stem cells from the dentate gyrus in the hippocampus and the subventricular zone of adult mice. Cultured adult neural stem cells are an important in vitro model to investigate stem cell properties such as proliferation and differentiation and to expand the understanding of plasticity in the adult brain. For complete details on the use and execution of this protocol, please refer to Isaksen et al. (2020).CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020).Microglia are the primary innate immune effectors of the central nervous system. Although numerous protocols have been developed to isolate fetal mouse microglia, the isolation of adult mouse microglia has proven more difficult. Here, we present a simple, widely accessible protocol to isolate pure microglia cultures from 4- to 14-month-old mouse brains using their adherent properties in vitro. These isolated microglia recapitulate the adherent properties of adult human microglia and present a more suitable model for studying age-related diseases. For complete details on the use and execution of this protocol in adult human microglia, please refer to Rustenhoven et al. (2016).This protocol describes how to prepare intact mouse cochleae for serial block-face scanning electron microscopy (SBEM). The detailed workflow includes cochlea fixation, en bloc staining, resin embedding, X-ray microscopy-guided trimming and SBEM data acquisition. This protocol allows large-scale, nanometer-resolution three-dimensional imaging of subcellular structures in a targeted tonotopic range of the cochlea and enables fast volumetric scan at submicron resolution using a compact X-ray microscope. For complete details on the use and execution of this protocol, please refer to Hua et al. (2021).Chromosome conformation capture (Hi-C) has become a routine method for probing the 3D organization of genomes. However, when applied to bacteria and archaea, current protocols are expensive and limited in their resolution. By dissecting the different steps of published eukaryotic and prokaryotic Hi-C protocols, we have developed a cost- and time-effective approach to generate high-resolution (down to 500 bp - 1 kb) contact matrices of both bacteria and archaea genomes. For complete details on the use and execution of this protocol, please refer to Cockram et al. (2020).Maternally and transiently accumulated SpCas9 (maternal SpCas9) in a zygote derived from a systemically SpCas9-expressing transgenic mouse strain was used to generate single- and multiple-gene-modified mice. Maternal SpCas9-based gene editing allows for high indel and knockin mutation efficiency, low mosaicism, increased pup delivery rate, and simultaneous induction of mutations at multiple loci in contrast to conventional CRISPR/SpCas9-based gene editing. For complete details on the use and execution of this protocol, please refer to Sakurai et al. (2020).Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. Selleck Dynasore By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019).The clinical effectiveness of BH3 mimetics therapy is limited by the inevitable emergence of acquired resistance. We present a protocol to model in vivo acquired resistance to BH3 mimetics in patient-derived xenograft (PDX) mouse models of acute myeloid leukemia. Using resistant PDXs as a valuable model, we next introduce a protocol for dynamic BH3 profiling (DBP) method. DBP allows functional identification of effective drug therapies based on measurements of drug-induced apoptosis signaling to overcome in vivo BH3 mimetics resistance. For complete details on the use and execution of this protocol, please refer to Bhatt et al. (2020).
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