Showing papers in "Methods in Cell Biology in 2012"
TL;DR: This tutorial introduces the lattice-based Glazier-Graner-Hogeweg (GGH) Monte Carlo multi-cell modeling and the open-source GGH-based CompuCell3D simulation environment that allows rapid and intuitive modeling and simulation of cellular and multi- cellular behaviors in the context of tissue formation and subsequent dynamics.
Abstract: The study of how cells interact to produce tissue development, homeostasis, or diseases was, until recently, almost purely experimental. Now, multi-cell computer simulation methods, ranging from relatively simple cellular automata to complex immersed-boundary and finite-element mechanistic models, allow in silico study of multi-cell phenomena at the tissue scale based on biologically observed cell behaviors and interactions such as movement, adhesion, growth, death, mitosis, secretion of chemicals, chemotaxis, etc. This tutorial introduces the lattice-based Glazier–Graner–Hogeweg (GGH) Monte Carlo multi-cell modeling and the open-source GGH-based CompuCell3D simulation environment that allows rapid and intuitive modeling and simulation of cellular and multi-cellular behaviors in the context of tissue formation and subsequent dynamics. We also present a walkthrough of four biological models and their associated simulations that demonstrate the capabilities of the GGH and CompuCell3D.
416 citations
TL;DR: The use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells are discussed and some minimally modified fluorophore-labeled sterols are described.
Abstract: Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.
203 citations
TL;DR: A correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections, is presented.
Abstract: The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions
128 citations
TL;DR: The implementation and application of currently used methods for target edoptical disruption in C. elegans are reviewed.
Abstract: Laser killing of cell nuclei has long been a powerful means of examining the roles of individual cells in C. elegans. Advances in genetics, laser technology, and imaging have further expanded the capabilities and usefulness of laser surgery. Here, we review the implementation and application of currently used methods for target edoptical disruption in C. elegans.
124 citations
TL;DR: This chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other TetrahYmena species.
Abstract: The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T thermophila and other Tetrahymena species Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented
111 citations
TL;DR: An explanation of how to appropriately choose between physical formulations that implicitly or explicitly account for cell geometry and between deterministic versus stochastic formulations for molecular dynamics is provided, along with a discussion of their respective strengths and weaknesses.
Abstract: The shape of a cell, the sizes of subcellular compartments and the spatial distribution of molecules within the cytoplasm can all control how molecules interact to produce a cellular behavior. This chapter describes how these spatial features can be included in mechanistic mathematical models of cell signaling. The Virtual Cell computational modeling and simulation software is used to illustrate the considerations required to build a spatial model. An explanation of how to appropriately choose between physical formulations that implicitly or explicitly account for cell geometry and between deterministic vs, stochastic formulations for molecular dynamics is provided, along with a discussion of their respective strengths and weaknesses. As a first step toward constructing a spatial model, the geometry needs to be specified and associated with the molecules, reactions and membrane flux processes of the network. Initial conditions, diffusion coefficients, velocities and boundary conditions complete the specifications required to define the mathematics of the model. The numerical methods used to solve reaction-diffusion problems both deterministically and stochastically are then described and some guidance is provided in how to set up and run simulations. A study of cAMP signaling in neurons ends the chapter, providing an example of the insights that can be gained in interpreting experimental results through the application of spatial modeling.
109 citations
TL;DR: This chapter highlights new methods in specimen preparation, imaging, and data analysis for accurate three-dimensional information can now be obtained for the whole animal at all stages, down to the level of individual organelles and the cytoskeleton.
Abstract: From its inception as a model organism 40 years ago, Caenorhabditis elegans was chosen in part for its suitability for study in serial thin sections by electron microscopy. Recent improvements in electron microscopy technology are making this pursuit more reliable and more powerful. In this chapter, we highlight new methods in specimen preparation, imaging, and data analysis. Accurate three-dimensional information can now be obtained for the whole animal at all stages, down to the level of individual organelles and the cytoskeleton.
106 citations
TL;DR: Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways, and new experimental tools are described that will facilitate a more comprehensive understanding of the DNA damage response.
Abstract: In response to genotoxic insults, cells activate DNA damage response pathways that either stimulate transient cell cycle arrest and DNA repair or induce apoptosis. The Caenorhabditis elegans germ line is now well established as a model system to study these processes in a genetically tractable, multicellular organism. Upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late-pachytene stage undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to apoptosis of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptotic machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we briefly review the C. elegans DNA damage response field, discuss in detail methods currently used to assay DNA damage responses in C. elegans, and describe the development of new experimental tools that will facilitate a more comprehensive understanding of the DNA damage response.
94 citations
TL;DR: An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described, which has the potential to extract statistically relevant data of structural details for systems biology.
Abstract: The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.
69 citations
TL;DR: An overview of common methods used to measure forces in both 2D and 3D microenvironments is provided and specific focus will be placed on traction force microscopy, which measures the force exerted by cells on 2D planar substrates, and the use of confocal reflectance microscope, which can be used to quantify collagen fibril compaction as a metric for 3D traction forces.
Abstract: Contractile force generation plays a critical role in cell adhesion, migration, and extracellular matrix reorganization in both 2D and 3D environments. Characterization of cellular forces has led to a greater understanding of cell migration, cellular mechanosensing, tissue formation, and disease progression. Methods to characterize cellular traction stresses now date back over 30 years, and they have matured from qualitative comparisons of cell-mediated substrate movements to high-resolution, highly quantitative measures of cellular force. Here, we will provide an overview of common methods used to measure forces in both 2D and 3D microenvironments. Specific focus will be placed on traction force microscopy, which measures the force exerted by cells on 2D planar substrates, and the use of confocal reflectance microscopy, which can be used to quantify collagen fibril compaction as a metric for 3D traction forces. In addition to providing experimental methods to analyze cellular forces, we discuss the application of these techniques to a large range of biomedical problems and some of the significant challenges that still remain in this field.
65 citations
TL;DR: A review of the current generation of genetic labels for direct protein identification by correlated light and electron microscopic (CLEM) imaging is presented in this paper, addressing their relative strengths and weaknesses and in what experiments they would be most useful.
Abstract: Correlated light and electron microscopic (CLEM) imaging is a powerful method for dissecting cell and tissue function at high resolution. Each imaging mode provides unique information, and the combination of the two can contribute to a better understanding of the spatiotemporal patterns of protein expression, trafficking, and function. Critical to these methods is the use of genetically appended tags that highlight specific proteins of interest in order to be able to pick them out of their complex cellular environment. Here we review and discuss the current generation of genetic labels for direct protein identification by CLEM, addressing their relative strengths and weaknesses and in what experiments they would be most useful.
TL;DR: This work presents a workflow that integrates different approaches: (1) correlative cryo-fluorescence microscopy to localize features within frozen-hydrated cells, (2) focused ion beam milling to thin these specimens in a targeted manner, and (3) cryo -electron tomography to provide detailed information about the cellular ultrastructure of thinned samples.
Abstract: The application of cryo-electron tomography to cells and tissues is commonly referred to as "cellular tomography," and enables visualization of the supramolecular architecture of cells in a near-native state. However, in order to access structural features hidden deep inside cellular volumes, it is necessary to use hybrid techniques to identify and localize features of interest and prepare such regions for subsequent analysis by transmission electron microscopy. We present a workflow that integrates different approaches: (1) correlative cryo-fluorescence microscopy to localize features within frozen-hydrated cells, (2) focused ion beam milling to thin these specimens in a targeted manner, and (3) cryo-electron tomography to provide detailed information about the cellular ultrastructure of thinned samples. We describe the combined use of these techniques and the instrumentation required to enable cryo-electron tomography for a vast range of cellular samples.
TL;DR: Although the MICs and MACs develop from mitotic products of a common progenitor and reside in a common cytoplasm, they are different from one another in almost every respect.
Abstract: Nuclear dualism is a characteristic feature of the ciliated protozoa. Tetrahymena have two different nuclei in each cell. The larger, polyploid, somatic macronucleus (MAC) is the site of transcriptional activity in the vegetatively growing cell. The smaller, diploid micronucleus (MIC) is transcriptionally inactive in vegetative cells, but is transcriptionally active in mating cells and responsible for the genetic continuity during sexual reproduction. Although the MICs and MACs develop from mitotic products of a common progenitor and reside in a common cytoplasm, they are different from one another in almost every respect.
TL;DR: Experimental data is reviewed exploring the developmental dependencies associated with both vegetative and conjugal development in ciliate Tetrahymena thermophila.
Abstract: The ciliate Tetrahymena thermophila can be said to undergo a variety of developmental programs. During vegetative growth, cells coordinate a variety of cell-cycle operations including macronuclear DNA synthesis and a-mitotic fission, micronuclear DNA synthesis and mitosis, cytokinesis and an elaborate program of cortical morphogenesis that replicates the cortical organelles. When starved, cells undergo oral replacement, transformation into fast-swimming dispersal forms or, when encountering cells of a complementary mating type, conjugation. Conjugation involves a 12 hour program of meiosis, mitosis, nuclear exchange and karyogamy, and two postzygotic divisions of the fertilization nucleus. This chapter reviews experimental data exploring the developmental dependencies associated with both vegetative and conjugal development.
TL;DR: This chapter describes detailed protocols for performing life span analysis in solid and liquid medium and describes various assays for measuring age-related changes in Caenorhabditis elegans.
Abstract: This chapter is dedicated to the study of aging in Caenorhabditis elegans (C. elegans). The assays are divided into two sections. In the first section, we describe detailed protocols for performing life span analysis in solid and liquid medium. In the second section, we describe various assays for measuring age-related changes. Our laboratory has been involved in several fruitful collaborations with non-C. elegans researchers keen on testing a role for their favorite gene in modulating aging (Carrano et al., 2009; Dong et al., 2007; Raices et al., 2008; Wolff et al., 2006). But even with the guidance of trained worm biologists, this undertaking can be daunting. We hope that this chapter will serve as a worthy compendium for those researchers who may or may not have immediate access to laboratories studying C. elegans.
TL;DR: Progress in CLEM is reviewed, with focus on matching the areas between different microscopic modalities, and a method that includes a virtual overlay and automated large-scale imaging, allowing to switch between most microscopes is introduced.
Abstract: Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals selected biomolecules or organelles but not the (ultra)structural context, as can be examined by electron microscopy (EM). LM and EM of the same cells, so-called correlative (or correlated) light and electron microscopy (CLEM), allow examining rare or dynamic events first by LM, and subsequently by EM. Here, we review progress in CLEM, with focus on matching the areas between different microscopic modalities. Moreover, we introduce a method that includes a virtual overlay and automated large-scale imaging, allowing to switch between most microscopes. Ongoing developments will revolutionize and standardize CLEM in the near future, which thus holds great promise to become a routine technique in cell biology.
TL;DR: This review starts with a description of the elaborate cortical organization of the Tetrahymena cell, and then proceeds inward to consider the mitochondria and then the nuclei, and includes a molecular inventory of the organelle or organelle system.
Abstract: Tetrahymena thermophila is both a cell and an organism, which combines great intracellular complexity with a remarkable accessibility to investigation using many different approaches. In this review, we start with a description of the elaborate cortical organization of the Tetrahymena cell, and then proceed inward to consider the mitochondria and then the nuclei. For each of these cellular organelles and organelle-systems, first we familiarize the reader with its location in the cell and its structure and ultrastructure, and then we analyze the molecular mechanisms associated with organelle assembly, function, and subdivision. This analysis includes a molecular inventory of the organelle or organelle system, as well as a review of the consequences of modification, disruption or overexpression of important molecular components of each structure or system. Relevant comparisons to results obtained with other well-studied organisms, from Paramecium to Homo sapiens, are also included. Our goal is to provide investigators, in particular those who are new to this organism, both the background and the motivation to work with this model system and achieve further insight into its organization and dynamics.
TL;DR: The development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.
Abstract: In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.
TL;DR: This chapter describes approaches for learning models of subcellular organization from images and the primary utility of these models is expected to be from incorporation into complex simulations of cell behaviors.
Abstract: This chapter describes approaches for learning models of subcellular organization from images. The primary utility of these models is expected to be from incorporation into complex simulations of cell behaviors. Most current cell simulations do not consider spatial organization of proteins at all, or treat each organelle type as a single, idealized compartment. The ability to build generative models for all proteins in a proteome and use them for spatially accurate simulations is expected to improve the accuracy of models of cell behaviors. A second use, of potentially equal importance, is expected to be in testing and comparing software for analyzing cell images. The complexity and sophistication of algorithms used in cell-image-based screens and assays (variously referred to as high-content screening, high-content analysis, or high-throughput microscopy) is continuously increasing, and generative models can be used to produce images for testing these algorithms in which the expected answer is known.
TL;DR: Nuclear small subunit rRNA and mitochondrial cytochrome c oxidase subunit 1 sequences indicate two major clades, a "borealis" clade of less closely related species and an "australis" clades of more closely relatedspecies that correlate to differences in mating-type determination and frequency of amicronucleates.
Abstract: The genus Tetrahymena is defined on the basis of a four-part oral structure composed of an undulating membrane and three membranelles. It is a monophyletic genus with 41 named species and numerous unnamed species, many of which are morphologically indistinguishable. Nuclear small subunit rRNA and mitochondrial cytochrome c oxidase subunit 1 sequences indicate two major clades, a "borealis" clade of less closely related species and an "australis" clade of more closely related species that correlate to differences in mating-type determination and frequency of amicronucleates. Members of both clades show convergence for histophagy (primarily facultative), macrostome transformation, and (rare) cyst formation. Life cycle parameters of species are presented and problematic species discussed.
TL;DR: A modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate, which drastically reduces the time needed to perform the test, minimizes damage to adherent cells, and decreases the possibility of losing floating cells.
Abstract: Analyzing apoptosis has been an integral component of many biological studies. Here we describe a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method is very simple and combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification. By eliminating the detaching and washing steps, our method drastically reduces the time needed to perform the test, minimizes damage to adherent cells, and decreases the possibility of losing floating cells.
TL;DR: This chapter describes how to process living specimens from the confocal microscope to the transmission electron microscopy (TEM), focusing on an improved ultramicrotomy technique that allows a precise and reliable targeting of the object of interest.
Abstract: Correlative light and electron microscopy (CLEM) is used when one needs to combine both imaging modalities on the same sample. When working on living small model organisms, such as Caenorhabditis elegans, specific CLEM protocols are required to acquire high-resolution light microscopic images of a region of interest and thereafter to relocate and study the same object at the ultrastructural level using a transmission electron microscope. In this chapter, we describe how to process living specimens from the confocal microscope to the transmission electron microscopy (TEM), focusing on an improved ultramicrotomy technique that allows a precise and reliable targeting of the object of interest. This improvement significantly reduces the time consuming and frequently frustrating search for the region of interest. Our targeted ultramicrotomy protocol is versatile enough to be applied on a variety of bulk specimens, such as fly and fish embryos, or mouse tissues.
TL;DR: A broad overview of patch-clamp analysis of neurons, muscles, and synapses in C. elegans can be found in this article, where the authors provide a methodological introduction to the techniques for patch clamping neurons and body-wall muscles in vivo.
Abstract: Patch-clamp electrophysiology is a technique of choice for the biophysical analysis of the function of nerve, muscle, and synapse in Caenorhabditis elegans nematodes. Considerable technical progress has been made in C. elegans electrophysiology in the decade since the initial publication of this technique. Today, most, if not all, electrophysiological studies that can be done in larger animal preparations can also be done in C. elegans. This chapter has two main goals. The first is to present to a broad audience the many techniques available for patch-clamp analysis of neurons, muscles, and synapses in C. elegans. The second is to provide a methodological introduction to the techniques for patch clamping C. elegans neurons and body-wall muscles in vivo, including emerging methods for optogenetic stimulation coupled with postsynaptic recording. We also present samples of the cell-intrinsic and postsynaptic ionic currents that can be measured in C. elegans nerves and muscles.
TL;DR: Detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans are presented.
Abstract: The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans.
TL;DR: Tetrahymena is a facile system with which to answer many biological questions, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation.
Abstract: Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions.
TL;DR: The rationale as well as advantages and potential pitfalls of methods used thus far to study metabolism and fat regulation, specifically triglyceride metabolism, in C. elegans are discussed.
Abstract: In Caenorhabdatis elegans as in other animals, fat regulation reflects the outcome of behavioral, physiological, and metabolic processes. The amenability of C. elegans to experimentation has led to utilization of this organism for elucidating the complex homeostatic mechanisms that underlie energy balance in intact organisms. The optical advantages of C. elegans further offer the possibility of studying cell biological mechanisms of fat uptake, transport, storage, and utilization, perhaps in real time. Here, we discuss the rationale as well as advantages and potential pitfalls of methods used thus far to study metabolism and fat regulation, specifically triglyceride metabolism, in C. elegans. We provide detailed methods for visualization of fat depots in fixed animals using histochemical stains and in live animals by vital dyes. Protocols are provided and discussed for chloroform-based extraction of total lipids from C. elegans homogenates used to assess total triglyceride or phospholipid content by methods such as thin-layer chromatography or used to obtain fatty acid profiles by methods such as gas chromatography/mass spectrometry. Additionally, protocols are provided for the determination of rates of intestinal fatty acid uptake and fatty acid breakdown by β-oxidation. Finally, we discuss methods for determining rates of de novo fat synthesis and Raman scattering approaches that have recently been employed to investigate C. elegans lipids without reliance on invasive techniques. As the C. elegans fat field is relatively new, we anticipate that the indicated methods will likely be improved upon and expanded as additional researchers enter this field.
TL;DR: This work focuses on the detection of strand breaks and oxidized bases in human peripheral blood mononuclear cells or cultured mammalian cells and covers preparation of cells, precoating of slides, embedding the cells in agarose, lysis, enzyme digestion, alkaline electrophoresis, fixation and staining, and scoring of comets.
Abstract: The comet assay (single-cell gel electrophoresis) is a simple, sensitive method for measuring DNA strand breaks, widely used in genotoxicity testing, human biomonitoring, ecogenotoxicology and fundamental research into mechanisms of DNA damage and repair. Cells embedded in agarose on a glass slide are lysed, leaving supercoiled DNA loops attached to the nuclear matrix as “nucleoids”. Electophoresis attracts DNA to the anode, but only those loops with breaks migrate, forming a comet-like image on fluorescence microscopy. The relative intensity of the comet tail reflects the frequency of DNA breaks, with a detection range up to a few thousand breaks per cell. DNA breaks, being produced by many diverse agents and as intermediates in DNA repair, are an unspecific marker of damage. More detailed information is obtained by incorporating a digestion with a lesion-specific endonuclease, after the lysis step. Here, we concentrate on the detection of strand breaks and oxidized bases in human peripheral blood mononuclear cells or cultured mammalian cells. We cover preparation of cells, precoating of slides, embedding the cells in agarose, lysis, enzyme digestion, alkaline electrophoresis, fixation and staining, and scoring of comets both visually and with computerized image analysis.
TL;DR: Fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section are described and fluorescent signals can be directly correlated to the corresponding subcellular structures in the area of interest.
Abstract: Correlative microscopy combines the versatility of the light microscope with the excellent spatial resolution of the electron microscope. Here, we describe fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section. The protocols are demonstrated on sections of tissue samples embedded in the methacrylate Lowicryl K4M. Ultrathin sections are mounted on electron microscopy (EM) grids and stained simultaneously with fluorescent and gold markers. For the detection of primary antibodies, we applied either protein A gold or immunoglobulin G (IgG) gold in combination with secondary antibodies coupled to Alexa488 or Alexa555. Alternatively, the correlative marker FluoroNanogold was used, followed by silver enhancement. The samples have to be analyzed first at the light microscope and then in the transmission electron microscope (TEM), because the fluorescence is bleached by the electron beam. Labeled structures selected at the fluorescence microscope can be identified in the TEM and analyzed at high resolution. This way, fluorescent signals can be directly correlated to the corresponding subcellular structures in the area of interest.
TL;DR: Results suggest that fibrin accumulation may not only increase the structural integrity of the thrombus but also considerably contribute toward limiting its growth.
Abstract: A multiscale computational model of thrombus (blood clot) development is extended by incorporating a submodel describing formation of fibrin network through "fibrin elements" representing regions occupied by polymerized fibrin. Simulations demonstrate that fibrin accumulates on the surface of the thrombus and that fibrin network limits growth by reducing thrombin concentrations on the thrombus surface and decreasing adhesivity of resting platelets in blood near thrombus surface. These results suggest that fibrin accumulation may not only increase the structural integrity of the thrombus but also considerably contribute toward limiting its growth. Also, a fast Graphics Processing Unit implementation is described for a multiscale computational model of the platelet-blood flow interaction.