Abstract:
:Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Baxter AE,Niessl J,Fromentin R,Richard J,Porichis F,Massanella M,Brassard N,Alsahafi N,Routy JP,Finzi A,Chomont N,Kaufmann DEdoi
10.1038/nprot.2017.079subject
Has Abstractpub_date
2017-10-01 00:00:00pages
2029-2049issue
10eissn
1754-2189issn
1750-2799pii
nprot.2017.079journal_volume
12pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::This protocol details a subset of assays developed within the touchscreen platform to measure various aspects of executive function in rodents. Three main procedures are included: extinction, measuring the rate and extent of curtailing a response that was previously, but is no longer, associated with reward; reversal ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.123
更新日期:2013-10-01 00:00:00
abstract::Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.191
更新日期:2007-01-01 00:00:00
abstract::Carbon-based dots (CDs) and their functionalized (nano)composites have recently attracted attention due to their seemingly easy preparation and numerous potential applications, ranging from those in the biomedical field (i.e., imaging and drug delivery) to those in (opto)electronics (i.e., solar cells and LEDs). This ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0207-x
更新日期:2019-10-01 00:00:00
abstract::Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the follow...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.147
更新日期:2016-12-01 00:00:00
abstract::This protocol details a method for obtaining a considerable number of human mast cells from peripheral blood cells from normal donors without using stem cell mobilization treatment. By using the magnetic cell sorting system, 10(4)-10(5) cells are retrieved in the CD34+ fraction when 100 ml of blood is drawn from a hea...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.344
更新日期:2006-01-01 00:00:00
abstract::Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0085-7
更新日期:2019-01-01 00:00:00
abstract::This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in a...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.270
更新日期:2006-01-01 00:00:00
abstract::The radial arm water maze (RAWM) contains six swim paths (arms) extending out of an open central area, with an escape platform located at the end of one arm (the goal arm). The goal arm location remains constant for a given mouse. On day 1, mice are trained for 15 trials (spaced over 3 h), with trials alternating betw...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.275
更新日期:2006-01-01 00:00:00
abstract::High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.436
更新日期:2012-01-05 00:00:00
abstract::The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiog...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.015
更新日期:2013-02-01 00:00:00
abstract::ErmineJ is software for the analysis of functionally interesting patterns in large gene lists drawn from gene expression profiling data or other high-throughput genomics studies. It can be used by biologists with no bioinformatics background to conduct sophisticated analyses of gene sets with multiple methods. It allo...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.78
更新日期:2010-06-01 00:00:00
abstract::This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.442
更新日期:2007-01-01 00:00:00
abstract::Over the past 20 years, the starlet sea anemone, Nematostella vectensis, a small estuarine animal, has emerged as a powerful model system for field and laboratory studies of development, evolution, genomics, molecular biology and toxicology. Here we describe how to collect Nematostella, culture it through its entire s...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.044
更新日期:2013-05-01 00:00:00
abstract::Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the s...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.017
更新日期:2015-02-01 00:00:00
abstract::Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into anima...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.078
更新日期:2017-09-01 00:00:00
abstract::Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundame...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.180
更新日期:2014-11-01 00:00:00
abstract::This protocol describes how to purify and radiolabel collagen for use as a substrate to assay collagenolytic members of the matrix metalloproteinases (MMPs). This assay measures enzymes that specifically cleave native triple helical collagen. After incubation of the MMP enzyme with the collagen substrate at 37 degrees...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.244
更新日期:2009-01-01 00:00:00
abstract::Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist i...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.238
更新日期:2010-03-01 00:00:00
abstract::The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defin...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.38
更新日期:2010-04-01 00:00:00
abstract::Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0237-4
更新日期:2019-12-01 00:00:00
abstract::The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocal...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0350-4
更新日期:2020-08-01 00:00:00
abstract::The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.313
更新日期:2011-04-01 00:00:00
abstract::At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA i...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.160
更新日期:2017-12-01 00:00:00
abstract::Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (m...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.018
更新日期:2015-02-01 00:00:00
abstract::Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD+ is one of the NCINs that has recently been discovered to cap RNAs in a wide range of species. Identification of the NAD+-capped RNAs (NAD-RNAs) could help to unveil the ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0363-z
更新日期:2020-09-01 00:00:00
abstract::The scototaxis (dark/light preference) protocol is a behavioral model for fish that is being validated to assess the antianxiety effects of pharmacological agents and the behavioral effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behavior. Briefly, a fish is placed in a centra...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.225
更新日期:2010-02-01 00:00:00
abstract::This protocol describes the principles and methods used for the preparation of cryopreserved cell stocks. Following these procedures will ensure the availability of reproducible cultures for use within a single laboratory at different times and for different collaborating laboratories. Although the basic principle is ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.190
更新日期:2008-01-01 00:00:00
abstract::Adult stem cell-based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-deriv...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0379-4
更新日期:2020-10-01 00:00:00
abstract::The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolisti...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.309
更新日期:2011-04-01 00:00:00
abstract::This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled micros...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.307
更新日期:2011-05-01 00:00:00