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Past Issue in 2020
Volume: 10, Issue: 22
Biochemistry
Total Triglyceride Quantification in Caenorhabditis elegans
Several studies suggest an important role of lipid metabolism in regulating longevity of Caenorhabditis elegans. Therefore, assays to quantify lipids have enormous value in understanding aging and pathologies associated with it. Approximately 70% of lipid metabolism genes in the nematode have orthologs in humans. Amenability of C. elegans to genetic manipulations has allowed investigations into the role of specific genetic factors in lipid metabolism. Here, we describe a protocol to quantify total triglycerides in C. elegans, which can be extended to studies of the effects of altered environmental and genetic factors on stored fats. This protocol quantifies the picomoles of the triglycerides, in whole worm lysate. Due to the sensitivity of the assay, it could help in identifying subtle changes in the total stored fat which are not discernible with microscopy techniques.
Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes
Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches. We have used this technique successfully to purify both GFP-tagged and mCherry-tagged proteins, and their interacting partners, expressed in Drosophila melanogaster embryos. Although we demonstrate the efficacy of the GFP-binding protein and RFP-binding protein nanobodies from Chromotek, in theory any antibody could be coupled to the beads and used as a Cl-AP reagent.
Biophysics
Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography
Electron cryotomography (cryo-ET) is an increasingly popular technique to study cellular structures and macromolecules in situ. Due to poor penetration of electrons through thick biological samples, the vitreously frozen samples for cryo-ET need to be thin. For frozen-hydrated cells, such samples can be produced either by cryomicrotomy or cryo-FIB-milling. As a result, a tomogram of such a sample contains information of a small fraction of the entire cell volume, making it challenging to image rare structures in the cell or to determine the distribution of scattered structures. Here, we describe the tools and workflow that we designed to facilitate serial cryomicrotomy, which makes possible the exploration of a larger volume of individual cells at molecular resolution. We successfully used serial cryomicrotomy to locate and image the Dam1/DASH complex located at microtubule plus ends inside mitotic Saccharomyces cerevisiae cells.
Cancer Biology
Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts
With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.
Magnet-assisted Flow Cytometry of in vivo Tumors to Quantitate Cell-specific Responses to Magnetic Iron Oxide Nanoparticles
A clear understanding of nanoparticle interactions with living systems at the cellular level is necessary for developing nanoparticle-based therapeutics. Magnetic iron oxide nanoparticles provide unique opportunities to study these interactions because of their responsiveness to magnetic fields. This enables sorting of cells containing nanoparticles from in vivo models. Once sorted, flow cytometry can identify individual cell types, which can be further analyzed for iron content, gene or protein expression changes associated with nanoparticle uptake, and for other biological responses at a molecular level. Here we provide a detailed protocol to sort and identify cells in the tumor microenvironment that have internalized magnetic iron oxide nanoparticles following intravenous administration.
Immunology
Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-host Interaction
Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.
Maternal Immune Activation with the Viral Mimetic Poly:IC in Pregnant Rats
Maternal immune activation (MIA) is increasingly well appreciated as an environmental risk factor for some psychiatric disorders. Administration of proinflammatory compounds such as the synthetic double-stranded RNA molecule polyinosinic-polycytidylic acid (polyI:C) to pregnant rodents results in the release of proinflammatory cytokines in the maternal circulation. Various behavioural and brain changes are produced in the offspring that are associated with psychiatric disorders such as autism and schizophrenia. This protocol describes the steps necessary for inducing MIA in pregnant rat dams, which will allow for investigations into the mechanisms in the dam and offspring that mediate the long-term effects of exposure to inflammation while in utero. Increasing our understanding of these mechanisms may provide new insights for the diagnosis, treatment, and prevention of psychiatric disorders. This protocol has been developed and improved over the years by various researchers in Dr. Howland’s laboratory at the University of Saskatchewan.
Microbiology
Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria
Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in liquid medium, we developed a simple and inexpensive peptide extraction procedure using a semi-solid nutrient-rich agar medium. We hereby present a detailed procedure that leads to the rapid extraction of secreted bioactive bacteriocin peptides from the oral species Streptococcus mutans, a prolific bacteriocin-producing species, and its potential application for bacteriocin extraction from other LAB (e.g., Streptococcus, Lactococcus, Enterococcus). We also present a simple method for the detection of bacteriocin activity from the purified extracellular peptide extract.
A Quick Method for Screening Biocontrol Efficacy of Bacterial Isolates against Bacterial Wilt Pathogen Ralstonia solanacearum in Tomato
Ralstonia solanacearum is a bacterial phytopathogen able to cause bacterial wilt disease in more than 200 plant species. Plant disease biocontrol strategies are used for controlling this disease and tomato is used as a model plant to conduct R. solanacearum associated studies. Conventional screening methods such as seed bacterization, soil drenching and root bacterization (in grown plants) to assess the ability of biocontrol bacteria to antagonize R. solanacearum under in planta conditions in different hosts are time-consuming and costly. A fast, cost effective method is a key requirement to advance the research on R. solanacearum biocontrol. In this protocol, we have inoculated the roots of tomato seedlings with bacterial isolates showing antagonistic activity against R. solanacearum under in vitro conditions. After 16 h of treatment with the antagonizing bacteria, seedlings were inoculated with R. solanacearum by a well-established root-dip method. Then the seedlings were maintained at controlled conditions and the number of wilted/dead seedlings were recorded up to 10th day post R. solanacearum inoculation. Biocontrol efficacy was calculated from the records for each tested isolate. This protocol is advantageous than already available protocols in the sense that it can be completed within a very short duration (~18 days for tomato) and there is no requirement of culture media to maintain the seedlings. This method can be used for quickly screening large number of bacterial isolates and different host genotypes within a short period of time and at a minimum cost.
Giant Mimiviridae CsCl Purification Protocol
While different giant viruses’ purification protocols are available, they are not fully described and they use sucrose gradient that does not reach an equilibrium. Here, we report a protocol for the purification of members of the Mimiviridae family virions resulting from Acanthamoeaba castellanii infections. Viruses are harvested after cell lysis and purified through a high density CsCl gradient to optimize the isolation of the virus from the cell debris or other potential contaminants. Due to the large size of the virion capsids, reaching half a micrometer diameter, the quality of the process can be monitored by light microscopy. The resulting purified particles can then be used to perform new infections, DNA extraction, structural studies, sugar composition analyses, sub-compartment characterization or proteomic experiments.
Neuroscience
In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts
Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then transduced with a plasmid(s) encoding a gene(s) of interest, aggregated by centrifugation, and cultured for 2 days in Matrigel. Time-lapse images of migratory behaviors of cultured neuroblasts and their ciliary structures, including the ciliary membrane and basal body, are acquired by confocal or superresolution laser-scanning microscopy. This method provides information about the spatiotemporal dynamics of neuroblasts’ morphology and ciliary structures, and is widely applicable to various types of migrating neuronal and nonneuronal cells in various species.
Visual-looming Shadow Task with in-vivo Calcium Activity Monitoring to Assess Defensive Behaviors in Mice
There has been a clear movement in recent years towards the adoption of more naturalistic experimental regimes for the study of behavior and its underlying neural architecture. Here we provide a protocol that allows experimenters working with mice, to mimic a looming and advancing predatory threat from the sky. This approach is easy to implement and can be combined with sophisticated neural recordings that allow access to real-time activity during behavior. This approach offers another option in a battery of tests that allow for a more comprehensive understanding of defensive behaviors.
Confocal Microscopy of Reovirus Transport in Living Dorsal Root Ganglion Neurons
Neurotropic reoviruses repurpose host machinery to traffic over long distances in neuronal processes and access distal replication sites. Understanding mechanisms of neuronal transmission is facilitated by using simplified in vitro primary neuronal culture models. Advances in the design of compartmentalized microfluidic devices lend robustness to neuronal culture models by enabling compartmentalization and manipulation of distinct neuronal processes. Here, we describe a streamlined methodology to culture sensory neurons dissociated from dorsal root ganglia of embryonic rats in microfluidic devices. We further describe protocols to exogenously label reovirus and image, track, and analyze transport of single reovirus particles in living neurons. These techniques can be adapted to study directed axonal transport of other neurotropic viruses and neuronal factors involved in signaling and pathology.
Plant Science
Agrobacterium-mediated Transformation of Sweet Basil (Ocimum basilicum)
Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we present a detailed protocol for a highly efficient Agrobacterium tumefaciens-mediated transformation method for sweet basil, which was established based on a previously reported method by other researchers, with modifications on several aspects, including growth of sweet basil, age of plants used for explants, preparation and concentration of Agrobacteria. This protocol allows researchers in academia and agroindustry to generate transgenic sweet basil plants in an easy, quick and highly reproducible manner. In addition, this protocol may be applicable to transform other species within the genus Ocimum.