Nature Protocols

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

Publication Date: 2012 PMID: 22596227
Authors: Wei, Q. X. - van der Hoeven, F. - Hollstein, M. - Odell, A. F.
Journal: Nat Protoc

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

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Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.

Publication Date: 2012 PMID: 22596226
Authors: Fridman, R. - Benton, G. - Aranoutova, I. - Kleinman, H. K. - Bonfil, R. D.
Journal: Nat Protoc

This protocol requires 2-4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 degrees C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.

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Mussel micronucleus cytome assay.

Publication Date: 2012 PMID: 22596225
Authors: Bolognesi, C. - Fenech, M.
Journal: Nat Protoc

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).

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Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy.

Publication Date: 2012 PMID: 22596224
Authors: Wu, S. - Baskin, T. I. - Gallagher, K. L.
Journal: Nat Protoc

Despite improvements in live imaging, fixation followed by embedding and sectioning for light or electron microscopy remains an indispensible approach in biology. During processing, small or delicate samples can be lost, damaged or poorly oriented. Here we present a protocol for overcoming these issues when, along with chemical fixation, the sample is fixed mechanically. The protocol features two alternatives for mechanical fixation: the sample is encased either in a rectangular block of agarose or between Formvar films suspended on a wire loop. We also provide methods for key steps all the way through to sectioning. We illustrate the method on the root of Arabidopsis thaliana, an object that is approximately 0.15 mm in diameter and difficult to process conventionally. With this protocol, well-oriented sections can be obtained with excellent ultrastructural preservation. The protocol takes about 1 week.

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A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

Publication Date: 2012 May PMID: 22582418
Authors: Diao, J. - Ishitsuka, Y. - Lee, H. - Joo, C. - Su, Z. - Syed, S. - Shin, Y. K. - Yoon, T. Y. - Ha, T.
Journal: Nat Protoc

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3-6 d.

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Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization.

Publication Date: 2012 Jun PMID: 22576108
Authors: Rao, H. - Tanpure, A. A. - Sawant, A. A. - Srivatsan, S. G.
Journal: Nat Protoc

This protocol describes the detailed experimental procedure for the synthesis of an azide-modified uridine triphosphate analog and its effective incorporation into an oligoribonucleotide by in vitro transcription reactions. Furthermore, procedures for labeling azide-modified oligoribonucleotides post-transcriptionally with biophysical probes by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and Staudinger reactions are also provided. This post-transcriptional chemical modification protocol is simple and modular, and it affords labeled oligonucleotides in reasonable amounts for biophysical assays. The procedure for enzymatic incorporation of the monophosphate of azide-modified UTP into an oligoribonucleotide transcript takes approximately 2 d, and subsequent post-transcriptional chemical functionalization of the transcript takes about 2 d.

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Visualization of gene expression in whole mouse retina by in situ hybridization.

Publication Date: 2012 Jun PMID: 22576107
Authors: Powner, M. B. - Vevis, K. - McKenzie, J. A. - Gandhi, P. - Jadeja, S. - Fruttiger, M.
Journal: Nat Protoc

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.

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Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells.

Publication Date: 2012 Jun PMID: 22576106
Authors: Zhang, J. - Nuebel, E. - Wisidagama, D. R. - Setoguchi, K. - Hong, J. S. - Van Horn, C. M. - Imam, S. S. - Vergnes, L. - Malone, C. S. - Koehler, C. M. - Teitell, M. A.
Journal: Nat Protoc

Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.

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Site-specific chemical protein conjugation using genetically encoded aldehyde tags.

Publication Date: 2012 Jun PMID: 22576105
Authors: Rabuka, D. - Rush, J. S. - Dehart, G. W. - Wu, P. - Bertozzi, C. R.
Journal: Nat Protoc

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with alpha-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

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Elucidating membrane structure and protein behavior using giant plasma membrane vesicles.

Publication Date: 2012 Jun PMID: 22555243
Authors: Sezgin, E. - Kaiser, H. J. - Baumgart, T. - Schwille, P. - Simons, K. - Levental, I.
Journal: Nat Protoc

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

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Genome-wide copy number analysis of single cells.

Publication Date: 2012 Jun PMID: 22555242
Authors: Baslan, T. - Kendall, J. - Rodgers, L. - Cox, H. - Riggs, M. - Stepansky, A. - Troge, J. - Ravi, K. - Esposito, D. - Lakshmi, B. - Wigler, M. - Navin, N. - Hicks, J.
Journal: Nat Protoc

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes approximately 3 d from flow cytometry to sequence-ready DNA libraries.

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Real-time detection of acetylcholine release from the human endocrine pancreas.

Publication Date: 2012 Jun PMID: 22555241
Authors: Rodriguez-Diaz, R. - Dando, R. - Huang, Y. A. - Berggren, P. O. - Roper, S. D. - Caicedo, A.
Journal: Nat Protoc

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes approximately 3 h.

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Using the rat forced swim test to assess antidepressant-like activity in rodents.

Publication Date: 2012 Jun PMID: 22555240
Authors: Slattery, D. A. - Cryan, J. F.
Journal: Nat Protoc

The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test involves the scoring of active (swimming and climbing) or passive (immobility) behavior when rodents are forced to swim in a cylinder from which there is no escape. There are two versions that are used, namely the traditional and modified FSTs, which differ in their experimental setup. For both versions, a pretest of 15 min (although a number of laboratories have used a 10-min pretest with success) is included, as this accentuates the different behaviors in the 5-min swim test following drug treatment. Reduction in passive behavior is interpreted as an antidepressant-like effect of the manipulation, provided it does not increase general locomotor activity, which could provide a false positive result in the FST.

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Video tracking and analysis of sleep in Drosophila melanogaster.

Publication Date: 2012 May PMID: 22538850
Authors: Gilestro, G. F.
Journal: Nat Protoc

In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of circadian behavior. However, proper analysis of sleep requires degrees of spatial and temporal resolution higher than is needed by circadian scientists, as well as different algorithms and software for data analysis. Here I describe how to perform sleep experiments in flies using techniques and software (pySolo and pySolo-Video) previously developed in my laboratory. I focus on computer-assisted video tracking to monitor fly activity. I explain how to plan a sleep analysis experiment that covers the basic aspects of sleep, how to prepare the necessary equipment and how to analyze the data. By using this protocol, a typical sleep analysis experiment can be completed in 5-7 d.

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Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP.

Publication Date: 2012 May PMID: 22538849
Authors: Bonn, S. - Zinzen, R. P. - Perez-Gonzalez, A. - Riddell, A. - Gavin, A. C. - Furlong, E. E.
Journal: Nat Protoc

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.

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Selective synthesis of 3-hydroxy acids from Meldrum's acids using SmI2-H2O.

Publication Date: 2012 May PMID: 22538848
Authors: Szostak, M. - Spain, M. - Procter, D. J.
Journal: Nat Protoc

The single-step synthesis of 3-hydroxy carboxylic acids from readily available Meldrum's acids involves a selective monoreduction using a SmI(2)-H(2)O complex to give products in high crude purity, and it represents a considerable advancement over other methods for the synthesis of 3-hydroxy acids. The protocol includes a detailed guide to the preparation of a single electron-reducing SmI(2)-H(2)O complex and describes two representative examples of the methodology: monoreduction of a fully saturated Meldrum's acid (5-(4-bromobenzyl)-2,2-dimethyl-1,3-dioxane-4,6-dione) and tandem conjugate reduction-selective monoreduction of alpha,beta-unsaturated Meldrum's acid (5-(4-methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione). The protocol for selective monoreduction of Meldrum's acids takes approximately 6 h to complete.

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Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts.

Publication Date: 2012 May PMID: 22538847
Authors: Shimamoto, Y. - Kapoor, T. M.
Journal: Nat Protoc

To explain how micrometer-sized cellular structures generate and respond to forces, we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based setup to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multimode imaging system allows the tracking of microtubules, chromosomes and needle tips. This setup can be used to analyze the viscoelastic properties of the spindle on timescales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 d.

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