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Past Issue in 2013
Volume: 3, Issue: 21
Biochemistry
Quantification of Bacterial Fatty Acids by Extraction and Methylation
This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty acids, both free and bound, from a bacterial culture, while the base derivatization protocol (Lennen and Pfleger, 2013) captures only bound (phospholipid, acyl-thioester) species. After extraction into hexane, the lipids may be analyzed by gas chromatography.
β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation.
Cell Biology
Ki67 Immunofluorescence on Bovine Cell Lines
This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).
Immunology
Bacterial Counts in Spleen
Bacterial loads can be determined as colony forming units (CFU) at any point of the infection by culturing spleen homogenates on agar plates. This is a reliable method for comparing the kinetics of infection in various mouse strains, estimating the virulence of different bacterial mutants or isolates and for vaccine testing and vacine estandarization. Although this method has been designed to recover Brucella or Salmonella organisms from spleen, the procedure may be applicable for other bacteria such as Listeria and Mycobacterium as well as to count bacterial loads in other organs such as liver or lymph nodes.
Microbiology
Immunoplaque Assay (Influenza Virus)
Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, genetic stability and purity of the virus population.
H2O2 Kill Assays of Biofilm Bacteria
Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized by marked tolerance to many biocides, including oxidants such as hydrogen peroxide. Hydrogen peroxide is both produced by host phagocytic cells, and used as an antimicrobial compound. Understanding biofilm tolerance to hydrogen peroxide is therefore relevant to the persistence of Pseudomonas aeruginosa in human infections (such as chronic Pseudomonas aeruginosa infections in cystic fibrosis airways) as well as in environmental settings (such as water pipes). This protocol was developed to determine the tolerance of Pseudomonas aeruginosa biofilms to hydrogen peroxide (H2O2) killing. The bacteria are grown as colony biofilms on polycarbonate membranes, as previously described in Walters et al. 2003. The protocol may be adapted for other bacterial, with appropriate changes in H2O2 concentrations, since different bacterial species may be more or less susceptible to H2O2 than Pseudomonas aeruginosa.
H2O2 Kill Assays of Planktonic Stationary Phase Bacteria
Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing of stationary phase bacterial cells to hydrogen peroxide and can be adapted to test other oxidants.
Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells
Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract mucosal epithelium. This protocol describes a method to measure adherence of M. catarrhalis to epithelial cell lines in vitro.
Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles
Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe a method to isolate and analyse M. catarrhalis fractions highly enriched for membrane proteins.
Neuroscience
Dissection of Different Areas from Mouse Hippocampus
The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the “hippocampus proper” which consists only of CA1, CA3 and smaller CA2 and CA4 areas, from the “hippocampal formation,” composed of these in addition to the dentate gyrus and subiculum). Distinct cell types give rise to unique axonal fiber pathways in the dentate gyrus, CA3 and CA1 subregions; accordingly, these areas may exhibit differential molecular profiles in response to a number of behavioral paradigms and pharmacological and genetic treatments. It is therefore in the interest of the investigator to dissect a specific subregion from the whole hippocampus. Here we outline a protocol for subregion-specific dissection from the adult mouse.
Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos
Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise developmental stages activating or blocking different signaling pathways. Here, we describe the manipulation of Wnt signaling pathway using Dkk-1-soaked micro-beads, implanted in ovo in the anterior part of the developing neural tube of chicken embryos; specifically at the prospective zona limitans intrathalamica at stage HH10.
Mitochondrial Isolation and Purification from Mouse Spinal Cord
Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of programmed cell death and that mitochondrial dysfunctions are implicated in numerous diseases including a wide range of neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. In order to identify and study changes in mitochondrial function related to specific neurological conditions the mitochondria are often isolated from the compartment of the central nervous system most affected during disease. Here, we describe a protocol for the isolation of mitochondria from mouse spinal cord, a compartment of the central nervous system that is significantly affected in neuromuscular diseases such as amyotrophic lateral sclerosis. This method relies on differential centrifugation to separate the mitochondria from the other subcellular compartments.
NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons
N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to acute brain insults such as stroke and brain trauma. Understanding the mechanisms underlying NMDAR-mediated excitotoxicity may lead to development of novel therapeutics for treating neurological diseases. Stimulation of primary cultured neurons with excessive NMDA is widely used as an in vitro model for studying NMDAR-mediated excitotoxicity, which allows careful dissection of the detailed cellular mechanisms underlying excitotoxic neuronal death.Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which can convert NAD into NADH. LDH is released from cells into culture medium when the plasma membrane integrity is compromised. Therefore, the amount of released LDH represents the degree of cell death. In our current study, the extracellular LDH level was measured using an in vitro Toxicology Assay Kit obtained from Sigma-Aldrich. The basis of this LDH assay is: 1) LDH reduces NAD into NADH, 2) the resulting NADH is then utilized in the stoichiometric conversion of a tetrazolium dye, and 3) the resulting colored compound is measured by a spectrophotometric microplate reader at a wavelength of 490 nm. The cell death rate was expressed as a percentage (%) between the absorbance of treated group and that of control group.
Plant Science
Ovule Clearing Method for Solanaceous Species
Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be particularly reliable for Solanaceous species.
Visualization and Quantification of Actin Dynamics in Rice Protoplasts
Direct visualization of the organization and dynamics of the actin cytoskeleton in rice cells is essential to understand its roles in regulating rice growth and development. Visualization of actin dynamics in protoplasts transformed with actin probe is a relatively quick and simple strategy, compared to the strategy of generating stable transgenic rice plants that harbor actin probe, which is time-consuming. Here is a protocol described in details regarding transforming rice protoplasts as well as visualizing and quantifying actin dynamics in rice protoplasts, which is based on the method previously reported (Shi et al., 2013).
Stem Cell
In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells
Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure of cell proliferation in adult murine hematopioetic stem cells (HSCs). Specifically, BrdU incorporation was analyzed for long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+) population.