Cold Spring Harbor Protocols

Publisher: Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press


Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is a definitive, interactive source of new and classic research techniques. The database is fully searchable by keyword and subject, and it has many novel features - such as discussion forums and personal folders - made possible by online publication. Its coverage includes cell and molecular biology, genetics, bioinformatics, protein science, and imaging. Protocols are presented step-by-step and edited in the style that has made Molecular Cloning, Antibodies, Cells and many other CSH manuals essential to the work of scientists worldwide. Protocols will be continuously expanded, updated, and annotated by the originators and users of the techniques. CSH Protocols - continuing Cold Spring Harbor Laboratory's 60-year tradition as a source of trusted techniques.

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Cold Spring Harbor Laboratory Press

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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Bioluminescence imaging (BLI) has become an essential technique for preclinical evaluation of anticancer therapeutics and provides sensitive and quantitative measurements of tumor burden in experimental cancer models. For light generation, a vector encoding firefly luciferase is introduced into human cancer cells that are grown as tumor xenografts in immunocompromised hosts, and the enzyme substrate luciferin is injected into the host. Alternatively, the reporter gene can be expressed in genetically engineered mouse models to determine the onset and progression of disease. In addition to expression of an ectopic luciferase enzyme, bioluminescence requires oxygen and ATP, thus only viable luciferase-expressing cells or tissues are capable of producing bioluminescence signals. Here, we summarize a BLI protocol that takes advantage of advances in hardware, especially the cooled charge-coupled device camera, to enable detection of bioluminescence in living animals with high sensitivity and a large dynamic range. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot078261.
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    ABSTRACT: Poisoned primer extension is primarily used to distinguish between RNAs that are nearly identical in sequence but cannot be distinguished by standard primer extension because they are the same size (e.g., edited vs. nonedited transcripts). It is conceptually identical to the standard primer extension reaction but involves the use of a chain-terminating dideoxynucleotide (the "poison") in the presence of the other three nucleotides. A radioactively labeled primer that hybridizes a short-distance downstream from the "changed" region of interest is extended by reverse transcription into this region of sequence variation. The reactions contain three of the four substrates for extension (e.g., dATP, dGTP, and dTTP) and a chain-terminating dideoxynucleotide (e.g., ddCTP). The extension reaction stops when reverse transcriptase adds a chain-terminating dideoxynucleotide to the template (e.g., it will add ddCTP when it encounters a G in the template sequence). RNAs that differ in sequence at that position will yield different-sized extension products that can be resolved on a denaturing gel. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot080986.
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    ABSTRACT: Initiator caspases, including caspase-2, -8, and -9, are activated by the proximity-driven dimerization that occurs after their recruitment to activation platforms. Here we describe the use of caspase bimolecular fluorescence complementation (caspase BiFC) to measure this induced proximity. BiFC assays rely on the use of a split fluorescent protein to identify protein-protein interactions in cells. When fused to interacting proteins, the fragments of the split fluorescent protein (which do not fluoresce on their own) can associate and fluoresce. In this protocol, we use the fluorescent protein Venus, a brighter and more photostable variant of yellow fluorescent protein (YFP), to detect the induced proximity of caspase-2. Plasmids encoding two fusion products (caspase-2 fused to either the amino- or carboxy-terminal halves of Venus) are transfected into cells. The cells are then treated with an activating (death) stimulus. The induced proximity (and subsequent activation) of caspase-2 in the cells is visualized as Venus fluorescence. The proportion of Venus-positive cells at a single time point can be determined using fluorescence microscopy. Alternatively, the increase in fluorescence intensity over time can be evaluated by time-lapse confocal microscopy. The caspase BiFC strategy described here should also work for other initiator caspases, such as caspase-8 or -9, as long as the correct controls are used. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot082552.
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    ABSTRACT: Chemical sequencing of RNA relies on the fact that each of the four bases in RNA is susceptible to chemical modification in a different way. In this protocol, end-labeled RNAs are subjected to base-specific chemical modification reactions that make the RNA strand adjacent to the modified base susceptible to cleavage. The chemical modification reaction is base-specific but limited so that not every base in every strand is modified. After cleavage, the resulting set of radioactive fragments is resolved via polyacrylamide gel electrophoresis. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot080937.
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    ABSTRACT: Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top083519.
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    ABSTRACT: The use of magnetic resonance imaging (MRI) in humans and animal models has been rapidly growing. MRI provides high spatial resolution, excellent tissue contrast, and outstanding definition of the anatomical structure of normal organs and tumors. Because MRI does not require genetically encoded reporters, it can be used for tumor surveillance and the assessment of treatment effects in a variety of mouse cancer models. MRI systems for preclinical imaging typically operate at higher magnetic field strength, ranging from 4.7 to 15 T, as opposed to clinical MRI scanners, which range from 1.5 to 3 T. The higher field strength of dedicated preclinical systems provides higher spatial resolution and higher signal-to-noise ratios. MRI of mouse cancer models requires optimization of numerous parameters, including pulse sequences and radio frequency coils. Here, we describe a protocol covering the general procedures for MRI. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot078253.
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    ABSTRACT: Genetically encoded, ratiometric, fluorescent Ca(2+) biosensors can be used in living cells to quantitatively measure free Ca(2+) concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca(2+) sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster resonance energy transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca(2+) concentrations. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot076547.
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    ABSTRACT: Conventional liquid chromatography on phosphocellulose (PC) can be used to separate tubulin and microtubule-associated proteins (MAPs). Tubulin is a highly acidic protein and thus does not bind to PC. MAPs, however, do bind to PC and can be eluted with a subsequent salt wash of the column. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot081208.
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    ABSTRACT: The field of single-cell analysis has greatly benefitted from recent technological advances allowing scientists to study genomes, transcriptomes, proteomes, and metabolomes at the single-cell level. Transcriptomics allows a unique window into cell function and is especially useful for studying global variability among single cells of seemingly the same type. Generating transcriptome data from RNA samples has become increasingly easy and can be done using either microarray or RNA-Seq techniques. RNA isolation is the first step of transcriptomics. Numerous RNA isolation procedures exist and differ with respect to the type and number of cells from which they are capable of isolating RNA. Although it is trivial to isolate RNA from bulk tissue or culture plates, sophisticated methods are required to capture RNA from single cells in a pool of cells or in intact tissue. We describe here the protocols used for isolating the soma of single neurons in cultures and in tissue slices using the pipette capture and the PALM or laser capture microdissection (LCM) approaches, respectively. LCM was developed to isolate cells from tissue sections primarily for pathological tissue analysis. LCM can be used to isolate individual cells or groups of cells from ethanol or paraffin-embedded formalin-fixed tissue sections and dissociated tissue cultures. The soma isolates from either technique can subsequently be used for RNA amplification procedures and transcriptome analysis. These procedures can also be adapted to other cell types in cultures and tissue sections and can be used on neuronal subcellular structures, such as dendrites. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot072439.
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    ABSTRACT: Caspases, a family of proteases that are essential mediators of apoptosis, are divided into two groups: initiator caspases and executioner caspases. Each initiator caspase is activated at the apex of its respective pathway, which generally leads to the cleavage and activation of executioner caspases. Executioner caspases in turn cleave numerous substrates in the cell, leading to its demise. Initiator caspases are activated when inactive monomers undergo induced proximity to form an active caspase. In contrast, executioner caspases are activated by cleavage. Based on this key difference, different imaging techniques have been developed to measure caspase activation and activity on a single-cell basis. Bimolecular fluorescence complementation (BiFC) is used to measure induced proximity of initiator caspases, whereas Förster resonance energy transfer (FRET) permits the investigation of caspase-mediated substrate cleavage in real time. Because many of the events in apoptosis, including caspase activation, are asynchronous in nature, these single-cell imaging techniques have proven to be immensely powerful in ordering and dissecting caspase pathways. When coupled with parallel detection of additional hallmark events of apoptosis, they provide detailed and quantitative kinetic and positional insights into the signal transduction pathways that regulate cell death. Here we provide a brief introduction into BiFC- and FRET-based imaging of caspase activation and activity in single cells. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top070342.
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    ABSTRACT: Förster resonance energy transfer (FRET) occurs across very short distances (in the nanometer range) between donor and acceptor fluorophores that overlap in their emission and absorption spectra. FRET-compatible green fluorescent protein (GFP) variants that are fused to short peptide linkers containing caspase cleavage sites can be used to measure caspase activity. In the intact probes, the donor and acceptor fluorophores are in close proximity, and FRET is highly efficient. On caspase activation, proteolysis of the linker occurs, and the donor is separated from the acceptor. This results in a disruption of resonance energy transfer and an increase in donor fluorescence quantum yield; this event is typically referred to as sensitized emission or donor unquenching. A number of highly sensitive FRET probes based on the cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair, or improved variants thereof, have been developed to detect intracellular caspase activities. In this protocol we describe how to use FRET-based caspase substrates and time-lapse imaging to measure caspase activity in cells undergoing apoptosis. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot082560.
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    ABSTRACT: In the last 15 years, there has been an explosion in the development of genetically encoded biosensors that report enzyme activity, chemical transformation, or concentration of ions and molecules in living cells. Currently, there are well over 120 biosensors of different cellular targets. As a general design principle, these sensors convert a molecular event, such as the binding of a molecule to a sensing domain or a signal-induced change in protein conformation, into a change in the sensor's fluorescence properties. In contrast to small-molecule sensors, genetically encoded sensors are generated when sensor-encoding nucleic acid sequences, which have been introduced by transgenic technologies, are translated in cells, tissues, or organisms. One of the best developed classes of biosensors is the genetically encoded Ca(2+) indicators (GECIs). Here, we briefly summarize the properties of ratiometric GECIs and describe how they are used to quantify Ca(2+) in specific cellular locations, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top066043.
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    ABSTRACT: Over the last two decades, advances in experimental and computational technologies have greatly facilitated genomic research. Next-generation sequencing technologies have made de novo sequencing of large genomes affordable, and powerful computational approaches have enabled accurate annotations of genomic DNA sequences. Charting functional regions in genomes must account for not only the coding sequences, but also noncoding RNAs, repetitive elements, chromatin states, epigenetic modifications, and gene regulatory elements. A mix of comparative genomics, high-throughput biological experiments, and machine learning approaches has played a major role in this truly global effort. Here we describe some of these approaches and provide an account of our current understanding of the complex landscape of the human genome. We also present overviews of different publicly available, large-scale experimental data sets and computational tools, which we hope will prove beneficial for researchers working with large and complex genomes. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.top083642.
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    ABSTRACT: The use of genetically encoded Ca(2+) sensors (GECIs) for long-term monitoring of intracellular Ca(2+) has become increasingly common in the last decade. Emission-ratiometric GECIs, such as those in the Yellow Cameleon family, can be used to make quantitative measurements, meaning that their fluorescence signals can be converted to free Ca(2+) concentrations ([Ca(2+)]free). This conversion is only as accurate as the sensor's apparent dissociation constant for Ca(2+) (K'd), which depends on temperature, pH, and salt concentration. This protocol describes a method for performing a titration, in living cells (in situ), of cytosolic, nuclear, or mitochondrial sensors. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot076554.
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    ABSTRACT: The microtubule-isolation procedures described here are based on the ability of the investigator to control the dimer-polymer distribution of tubulin by varying the temperature of the extract. In general, the extract is warmed to induce microtubule assembly, the polymer is collected by centrifugation, cooled to induce disassembly, clarified by centrifugation, and then warmed again to produce polymer. As long as the GTP supply is sufficient, the microtubules that result can be taken through numerous rounds of this in vitro assembly and disassembly reaction. Many microtubule-associated proteins (MAPs) associate with microtubules assembled in vitro. Some reagents can skew the equilibrium of assembly and disassembly toward formation of polymer. The inclusion of glycerol, for instance, promotes microtubule assembly by disrupting the hydration shell around the tubulin dimers. The result is a greater yield of tubulin per gram of starting material. However, the ratio of MAPs to tubulin is slightly lower, presumably because the glycerol also decreases the binding of MAPs to tubulin. Two methods are described here: The first uses buffer lacking assembly-promoting components, and the second uses buffer containing glycerol. These procedures are most efficient with vertebrate brain tissue, where the soluble protein can be up to 15%-20% tubulin. The first produces satisfactory yields when using chick or pig brain; the second is recommended for calf or cow brain. The second procedure may also be useful for studies of nonneuronal tissues where the relative concentration of tubulin per gram of wet weight is considerably lower than that of brain. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot081182.
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    ABSTRACT: Paclitaxel binds to tubulin, strongly promotes microtubule assembly from subunits, and stabilizes the assembled polymer against disassembly. Because of its ability to drive the assembly reaction almost completely toward microtubules, the paclitaxel-dependent procedure outlined here is particularly useful for the isolation of microtubules from tissues in which the intracellular concentration of tubulin is low (e.g., nonneuronal sources, cultured cells, and invertebrate tissues). The microtubule-associated proteins (MAPs) remain bound to the paclitaxel-stabilized microtubules. The isolation of these MAPs by salt extraction is also described here. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(1):pdb.prot081190.
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    ABSTRACT: The binding of a protein to an RNA sequence protects the region of the RNA from cleavage by chemicals or RNases; this protected region is known as the protein's "footprint." In the footprinting protocol presented here, end-labeled RNAs with and without bound protein are cleaved using chemical methods. Fe(II)-EDTA is used to generate hydroxyl radicals in the presence of a reducing agent. These hydroxyl radicals indiscriminately cleave ribose groups in regions of the ribose-phosphate backbone that are exposed to solvent. After termination of cleavage, the resulting RNA fragments are analyzed by gel electrophoresis on denaturing polyacrylamide gels. Because hydroxyl radicals are smaller and cleave less specifically than RNases, this approach, if feasible, is often the method of choice for monitoring sites of RNA-protein interactions. © 2014 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 12/2014; 2014(12):pdb.prot080952.
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    ABSTRACT: This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in Molecular Neuroscience (ATMN) lecture and laboratory course at Cold Spring Harbor Laboratory (CSHL) for nearly a decade. Lentiviruses are derived from HIV-1 and are ideal vehicles for the delivery of multiple genes of interest into target cells. In this protocol, 2A peptide-linked sequences are used to create a bicistronic lentiviral construct containing a ubiquitous promoter (chick β actin with a cytomegalovirus [CMV] early enhancer) driving dual expression of two fluorescent proteins (FP): H2B-Cerulean (a nuclear-localized blue FP) and Dendra2 (a photoactivatable green FP that converts to red after exposure to UV light). Polymerase chain reaction (PCR) amplification of the bicistronic insert is followed by subcloning into a lentiviral vector and transfection into a packaging cell line. The resulting viral supernatants can be used to prepare concentrated stocks and infect cells for imaging via epifluorescent and confocal microscopy. © 2014 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 12/2014; 2014(12):pdb.prot081422.
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    ABSTRACT: Purifying and culturing cells from the central nervous system (CNS) has proved to be an incredibly powerful tool for dissecting fundamental neuron and glial properties, and especially powerful in understanding neuronal-glial interactions. In a series of detailed protocols, we have provided step-by-step instructions for purifying and culturing specific types of neurons, glia, and vascular cells from the CNS by immunopanning. This article discusses common pitfalls and errors as well as important design considerations for the immunopanning procedure. © 2014 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 12/2014; 2014(12):pdb.ip073999.
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    ABSTRACT: This protocol describes the use of immunopanning for acute purification and primary culture of Schwann cells from intact neonatal and injured adult mouse sciatic nerve. © 2014 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 12/2014; 2014(12):pdb.prot074989.