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Nature Protocols

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https://www.readbyqxmd.com/read/29345636/promoting-the-accumulation-of-tumor-specific-t-cells-in-tumor-tissues-by-dendritic-cell-vaccines-and-chemokine-modulating-agents
#1
Nataša Obermajer, Julie Urban, Eva Wieckowski, Ravikumar Muthuswamy, Roshni Ravindranathan, David L Bartlett, Pawel Kalinski
This protocol describes how to induce large numbers of tumor-specific cytotoxic T cells (CTLs) in the spleens and lymph nodes of mice receiving dendritic cell (DC) vaccines and how to modulate tumor microenvironments (TMEs) to ensure effective homing of the vaccination-induced CTLs to tumor tissues. We also describe how to evaluate the numbers of tumor-specific CTLs within tumors. The protocol contains detailed information describing how to generate a specialized DC vaccine with augmented ability to induce tumor-specific CTLs...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29300391/use-of-a-three-layer-gradient-system-of-cells-for-rat-testicular-organoid-generation
#2
João Pedro Alves-Lopes, Olle Söder, Jan-Bernd Stukenborg
We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood-testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5-7 d in culture...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29300390/a-surgical-orthotopic-organoid-transplantation-approach-in-mice-to-visualize-and-study-colorectal-cancer-progression
#3
Arianna Fumagalli, Saskia J E Suijkerbuijk, Harry Begthel, Evelyne Beerling, Koen C Oost, Hugo J Snippert, Jacco van Rheenen, Jarno Drost
Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a large collection of tumor material faithfully recapitulating phenotypic and genetic heterogeneity of native tumors. To study tumor progression in the natural in vivo environment, we developed an orthotopic approach based on transplantation of CRC organoids into the cecal epithelium...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29300389/high-throughput-in-situ-x-ray-screening-of-and-data-collection-from-protein-crystals-at-room-temperature-and-under-cryogenic-conditions
#4
Jana Broecker, Takefumi Morizumi, Wei-Lin Ou, Viviane Klingel, Anling Kuo, David J Kissick, Andrii Ishchenko, Ming-Yue Lee, Shenglan Xu, Oleg Makarov, Vadim Cherezov, Craig M Ogata, Oliver P Ernst
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29300388/colonoscopy-based-colorectal-cancer-modeling-in-mice-with-crispr-cas9-genome-editing-and-organoid-transplantation
#5
Jatin Roper, Tuomas Tammela, Adam Akkad, Mohammad Almeqdadi, Sebastian B Santos, Tyler Jacks, Ömer H Yilmaz
Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations-such as the subcutaneous space, kidney capsule, or cecal wall-that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29323664/on-demand-synthesis-of-organozinc-halides-under-continuous-flow-conditions
#6
Mateo Berton, Lena Huck, Jesús Alcázar
Organozinc reagents are versatile building blocks for introducing C(sp2)-C(sp3) and C(sp3)-C(sp3) bonds into organic structures. However, despite their ample synthetic versatility and broad functional group tolerance, the use of organozinc reagents in the laboratory is limited because of their instability, exothermicity and water sensitivity, as well as their labor-intensive preparation. Herein, we describe an on-demand synthesis of these useful reagents under continuous flow conditions, overcoming these primary limitations and supporting widespread adoption of these reagents in synthetic organic chemistry...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29323663/multiplexed-proteome-analysis-with-neutron-encoded-stable-isotope-labeling-in-cells-and-mice
#7
Katherine A Overmyer, Stefka Tyanova, Alex S Hebert, Michael S Westphall, Jürgen Cox, Joshua J Coon
We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29323662/crispr-cas9-based-genome-wide-screening-of-toxoplasma-gondii
#8
Saima M Sidik, Diego Huet, Sebastian Lourido
Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein-coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29266099/3d-molecular-cartography-using-lc-ms-facilitated-by-optimus-and-ili-software
#9
Ivan Protsyuk, Alexey V Melnik, Louis-Felix Nothias, Luca Rappez, Prasad Phapale, Alexander A Aksenov, Amina Bouslimani, Sergey Ryazanov, Pieter C Dorrestein, Theodore Alexandrov
Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29266098/easi-crispr-for-creating-knock-in-and-conditional-knockout-mouse-models-using-long-ssdna-donors
#10
Hiromi Miura, Rolen M Quadros, Channabasavaiah B Gurumurthy, Masato Ohtsuka
CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29266097/single-cell-microscopy-of-suspension-cultures-using-a-microfluidics-assisted-cell-screening-platform
#11
Burak Okumus, Charles J Baker, Juan Carlos Arias-Castro, Ghee Chuan Lai, Emanuele Leoncini, Somenath Bakshi, Scott Luro, Dirk Landgraf, Johan Paulsson
Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29266096/live-cell-measurements-of-kinase-activity-in-single-cells-using-translocation-reporters
#12
Takamasa Kudo, Stevan Jeknić, Derek N Macklin, Sajia Akhter, Jacob J Hughey, Sergi Regot, Markus W Covert
Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29240734/transient-expression-of-human-antibodies-in-mammalian-cells
#13
Rodrigo Vazquez-Lombardi, Damien Nevoltris, Ansha Luthra, Peter Schofield, Carsten Zimmermann, Daniel Christ
Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29240733/use-of-tai-fish-to-visualize-neural-ensembles-activated-by-multiple-stimuli
#14
Qi Zhang, Qiye He, Jihua Wang, Chaoying Fu, Hailan Hu
Researchers in behavioral neuroscience have long sought imaging techniques that can identify and distinguish neural ensembles that are activated by sequentially applied stimuli at single-cell resolution across the whole brain. Taking advantage of the different kinetics of immediate-early genes' mRNA and protein expression, we addressed this problem by developing tyramide-amplified immunohistochemistry-fluorescence in situ hybridization (TAI-FISH), a dual-epoch neural-activity-dependent labeling protocol. Here we describe the step-by-step procedures for TAI-FISH on brain sections from mice that were sequentially stimulated by morphine (appetitive first stimulus) and foot shock (aversive second stimulus)...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29215635/mapping-the-small-rna-interactome-in-bacteria-using-ril-seq
#15
Sahar Melamed, Raya Faigenbaum-Romm, Asaf Peer, Niv Reiss, Omer Shechter, Amir Bar, Yael Altuvia, Liron Argaman, Hanah Margalit
Small RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. To enable transcriptome-wide mapping of bacterial sRNA-target pairs, we developed RIL-seq (RNA interaction by ligation and sequencing). RIL-seq is an experimental-computational methodology for capturing sRNA-target interactions in vivo that takes advantage of the mutual binding of the sRNA and target RNA molecules to the RNA chaperone protein Hfq. The experimental part of the protocol involves co-immunoprecipitation of Hfq and bound RNAs, ligation of RNAs, library preparation and sequencing...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29215634/expansion-of-patient-derived-circulating-tumor-cells-from-liquid-biopsies-using-a-ctc-microfluidic-culture-device
#16
Bee Luan Khoo, Gianluca Grenci, Ying Bena Lim, Soo Chin Lee, Jongyoon Han, Chwee Teck Lim
The development of personalized cancer therapy depends on a robust system to monitor the patient's individual response to anticancer treatment. Anticancer drug efficacy has been tested on circulating tumor cells (CTCs) derived from patient blood samples after ex vivo expansion into CTC clusters. Current attempts to culture these primary cancer cells focus on long-term maintenance under growth factor supplements into cell lines, which usually takes >6 months and results in a CTC expansion efficiency of <20%...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29215633/measuring-mutation-accumulation-in-single-human-adult-stem-cells-by-whole-genome-sequencing-of-organoid-cultures
#17
Myrthe Jager, Francis Blokzijl, Valentina Sasselli, Sander Boymans, Roel Janssen, Nicolle Besselink, Hans Clevers, Ruben van Boxtel, Edwin Cuppen
Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29215632/assembly-of-phospholipid-nanodiscs-of-controlled-size-for-structural-studies-of-membrane-proteins-by-nmr
#18
Franz Hagn, Mahmoud L Nasr, Gerhard Wagner
Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29189775/visualizing-endocytic-recycling-and-trafficking-in-live-neurons-by-subdiffractional-tracking-of-internalized-molecules
#19
Merja Joensuu, Ramon Martínez-Mármol, Pranesh Padmanabhan, Nick R Glass, Nela Durisic, Matthew Pelekanos, Mahdie Mollazade, Giuseppe Balistreri, Rumelo Amor, Justin J Cooper-White, Geoffrey J Goodhill, Frédéric A Meunier
Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29189774/deriving-genotypes-from-rad-seq-short-read-data-using-stacks
#20
Nicolas C Rochette, Julian M Catchen
Restriction site-associated DNA sequencing (RAD-seq) allows for the genome-wide discovery and genotyping of single-nucleotide polymorphisms in hundreds of individuals at a time in model and nonmodel species alike. However, converting short-read sequencing data into reliable genotype data remains a nontrivial task, especially as RAD-seq is used in systems that have very diverse genomic properties. Here, we present a protocol to analyze RAD-seq data using the Stacks pipeline. This protocol will be of use in areas such as ecology and population genetics...
December 2017: Nature Protocols
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