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Past Issue in 2016
Volume: 6, Issue: 10
Cell Biology
Isolating Liver Mitochondria by Differential Centrifugation
In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
Liquid Luminescent DNA-precipitation Assay
Working on transcription factors requires studying interactions between protein and DNA. After identification of putative binding-sequences and motifs, Electrophoretic Mobility Shift Assay (EMSA) experiment is classically used to determine specific interactions of proteins and nucleic acids. This lengthy process is rather heavy-handed because of radioisotopically labeled DNA and autoradiographic visualization that are required for the experiments.Liquid luminescent DNA precipitation assay provides rapid, reliable and quantitative results concerning protein-DNA interactions. This protein-DNA binding assay is based on solution hybridization between Digoxigenin-labeled (DIG) DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to Glutathione Sepharose 4B beads (Figure 1), without electrophoresis (Toshiharu et al., 2008). Digoxigenin is a steroid found in plants. It is increasingly used as a label for nonradioactive detection of nucleic acids and proteins.Figure 1. Representation of liquid chemiluminescent DNA pull-down assay. A Glutathione S-transferase (GST)-fused NLRP3 (GST-NLRP3) bound to Glutathione Sepharose 4B beads is incubated with a DIG-labeled double-stranded DNA fragment containing putative NLRP3 Binding Site (NBS) in protein-DNA binding buffer. After extensive washing, protein-DNA binding on beads is detected using anti-DIG antibody conjugated to alkaline phosphatase, which is measured by a chemiluminescent reaction using a luminometer Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13, 7] decan}-4-yl) phenyl phosphate(CSPD).Here, we described how we used this technique to demonstrate the interaction between NLRP3 protein and its DNA binding site (Bruchard et al., 2015).
Immunology
Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells
Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and solve this kind of issue. Moreover PLA experiments are read out using fluorescence or bright field microscopy, providing additional information on intracellular interactions localization significantly bettering classical IP procedures.PLA reagents are made of complementary small oligonucleotides “minus” and “plus” probes which specifically recognize host species from the primary antibodies (Abs) targeting the two proteins you are interested in. Experiments have to be designed with primary Abs from different species (rabbit, mouse or goat) as PLA probes “minus” or “plus” react against a specific host species of the primary antibody (e.g. “plus” anti-rabbit with “minus” anti-mouse or “plus” anti-mouse with “minus” anti-rabbit combos are allowed if mouse and rabbit primary Abs are used). When the two PLA probes are close enough (40 nm) a ligation occurs upon ligase incubation generating a circle DNA. These circle-forming DNAs are next amplified thanks to a polymerase and complementary fluorescent nucleotides, being incorporated at this step. Each luminescent spot is thereafter considered being an interaction site between the two proteins (Figure 1).Figure 1. Schematic representation of Duolink® experiment. Primary Abs from different host species were used [i.e., Mouse (Ms) anti-Nlrp3 and Rabbit (Rb) anti-IRF4]. When protein-protein interaction occurs PLA probes allow incorporation of fluorescent oligonucleotides which are analyzed by microscopy.PLA experiments have been performed on differentiated T cells from mice. T cells were grown on cover slip coated with poly-L-Lysine. Please be aware that for T cells and other non-adherent cells, experiments and staining are also possible in 500 µl microtubes until the last washing step before mounting cover slip on slide. This microtube method saves cytokines reagents and allows T cells to grow with usual methods but centrifugation repetition for washing steps is hazardous. Primary Abs incubation also requires also a specific setup if microtube method is chosen.
Extraction and Quantification of Sphingosine 1-Phosphate (S1P)
Sphingosine 1-phosphate (S1P) is a lipid metabolite and signaling molecule involved in many different physiological processes including lymphocyte circulation, T cell differentiation, antigen presentation, and maintenance of the vascular endothelial barrier. S1P is a ligand of five different G protein-coupled cell surface receptors designated S1P1-5. It has also been described as an intracellular second messenger. Quantification of S1P in biological samples is therefore an important task to decipher its signaling capabilities in vivo under physiological and pathophysiological conditions in different body fluids and organs. In this protocol, quantification of S1P is performed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS).
Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets
Transfer of mature T cells into immunodeficient mice results in sub-optimal reconstitution of the peripheral T cell pool. Under lymphopenic conditions, dendritic cells are released from tonic control by regulatory T cells (Tregs), and consequently drive activation and proliferation of low affinity T cells specific for endogenous antigens. This oligoclonal proliferation results in a T cell population dominated by T cells possessing an effector/memory phenotype and a limited TCR repertoire. Oligoclonal expansion can be prevented by selectively reconstituting the Treg compartment prior to T cell transfer (Bolton et al., 2015). Reconstitution of the Treg compartment of lymphopenic mice has been tested in immunodeficient mouse strains such as Rag-1-/-or Rag-2-/- mice, and in immunosufficient mice rendered transiently lymphopaenic by lethal whole body irradiation as conditioning for bone marrow transplantation (BMT). Transfer of purified Tregs into these hosts, combined with treatment with exogenous IL-2 for 7 days, is sufficient to reconstitute the Treg compartment and reduce expression of dendritic cell costimulatory molecules, a critical process in preventing inappropriate expansion of self-reactive T cells. T cells transferred after Treg reconstitution do not undergo rapid spontaneous proliferation, and instead undergo slow homeostatic division to repopulate the T cell pool with naive T cells, thus allowing optimal reconstitution of peripheral T cell pool.
Study of Epithelium Barrier Functions by Real-time TER Measurement
Transepithelial Electrical Resistance (TER) measurement is a reliable and efficient method to quantify the permeability of barrier forming cells such as epithelial cells. Measuring the permeability of the epithelial cells will help the researchers to investigate the barrier function of epithelium in various infectious and inflammatory diseases. Here we provide a real-time and impedance-based approach for measuring the permeability of epithelial cell monolayer using the Electrical Cell Substrate Impedance Sensing (ECIS®) instrumentation.
Microbiology
Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). Here we discuss in detail how to prepare RSV stocks with a high or low content of DVGs, and how to purify defective viral particles containing DVGs from an RSV stock enriched in defective viral particles. These procedures are useful for the preparation of viral stocks and defective viral particles necessary for laboratory research. In brief, the different RSV stocks are produced in HEp2 cells, which are commonly used to amplify this virus in the laboratory. To generate an RSV stock with a high content of DVGs, HEp2 cells are sequentially infected with a high multiplicity of infection (MOI) multiple times followed by purification of the viral particles containing DVGs using gradient centrifugation. The procedure describe here has four parts: 1. Amplification of seed RSV stock with a low DVG content (RSV-LD), 2. Generation of a stock with a high DVG content (RSV-HD), 3. Purification of DVGs by gradient centrifugation, 4. Characterization of purified DVGs.
Nitrite Reduction Assay for Whole Pseudomonas Cells
The second step of the dissimilatory denitrification pathway in which nitrite (NO2-) is converted to nitric oxide (NO) is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron (Fe), in the form of heme, or copper (Cu) (Zumft, 1997). The Pseudomonas stutzeri (P. stutzeri) RCH2 strain used in this assay contains both an Fe and a Cu form of nitrite reductase. In this assay, total nitrite reductase activity can be measured in whole cells using fumarate or some other carbon source as an electron source by measuring the disappearance of nitrite over time (Thorgersen et al., 2015).
Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR
Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and exacerbation of chronic lung conditions including asthma, and it is a major cause of hospitalizations in infants. Because of its clinical relevance, experimental animal models to study RSV in vivo are needed. The most common and accessible animal model in research laboratories is the mouse. However, commonly use RSV strains poorly establish infection in mice and thus titration of the virus from mouse lungs to confirm infection is not sensitive enough to detect early viral infection. Here we discuss in detail how to infect BALB/c mice with RSV and how to detect RSV genomes in the lung using reverse transcription quantitative PCR (RT-qPCR). This method allows detection of viral genomes as early as day 1 post-infection (shown in Figure 2), whereas traditional TCID50 fails to detect significant virus until after day 2 post-infection. Of note, despite of higher sensitivity, genome RT-qPCR only shows the production of viral genomes and thus positive results for this assay are not proof of production of infectious viral particles.
Induction, Isolation and Counting of Akinetes in Aphanizomenon ovalisporum
Akinetes are spore-like resting (dormant) cells formed by strains of filamentous cyanobacteria for surviving long periods of unfavorable conditions. During deprivation for potassium, vegetative photosynthetic cells along the filaments of the cyanobacterium Aphanizomenon ovalisporum (A. ovalisporum) (strain ILC-164) differentiate into akinetes. Akinetes are larger than vegetative cell, have a thick wall, accumulate storage compounds (cyanophycine, glycogen, lipids) and excess of DNA (Sukenik et al., 2015; Sukenik et al., 2007; Maldener et al., 2014). Differences in structure and composition between akinetes and vegetative cells allow separation and isolation of akinetes. Akinetes isolated by the described protocol can be utilized for protein analysis, measurements of metabolic activities, fluorescence in situ hybridization (FISH) studies and more.
Molecular Biology
Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex
To prepare the knockdown transformants of the Closterium peracerosum-strigosum-littorale (C. psl.) complex, particle bombardment was applied with a newly constructed vector (pSA0104) with an endogenous constitutive promoter fused to a DNA fragment corresponding to an antisense strand of a target gene. Using a hygromycin resistance gene (aph7”), hygromycin-resistant colonies were selected. After the second screening, integration of the vector into the genome was checked by PCR and the knockdown effect was evaluated by Western blotting using a specific antibody against the target protein.
Affymetrix Genome-wide Human SNP Assay
To assess genomic variation, it is possible to identify the single nucleotide polymorphisms (SNP) which an individual carries. Using the Affymetrix Genome-wide Human SNP Assay, it is possible to assess 906,600 SNPs on a single array. This protocol, the next iteration of the GeneChip Mapping 500K array set, is based directly on the manufacturers’ protocol and shows steps which are highly similar to that which is found here: http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf.
Neuroscience
Isolating Brain Mitochondria by Differential Centrifugation
In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice brain tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
Running Reward Conditioned Place Preference Task
The conditioned place preference (CPP) test is a standard pre-clinical behavioral tool used to study the motivational effects of drugs and non-drug treatments in experimental animals. The basic characteristic of this task involves the association of a particular environment and contextual cues with a reward stimulus, followed by the association of a different environment with the absence of the reward stimulus (Prus and Rosecrans, 2009). Besides the motor component, voluntary wheel running exercise also has a rewarding component, and has been suggested as a strong natural reinforcer. Consistent with this notion, rodents will readily begin to run when a wheel is introduced (Eikelboom and Mills, 1988; Looy and Eikelboom, 1989), will work by lever pressing to gain access to a running wheel (Pierce et al., 1986), and spend more time in a place previously associated with the aftereffects of running (Lett et al., 2000; Lett et al., 2001). More recently, we underscored an important role for the adipocyte-derived hormone leptin in midbrain dopamine neurons in the modulation of running reward (Fernandes et al., 2015). Here, we describe a CPP protocol to measure the rewarding aftereffects of wheel running exercise in mice.
Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons
This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the cell surface of live cultured neurons are biotinylated. Neuronal extracts are prepared and biotinylated proteins are collected with NeutrAvidin-coupled beads, and analyzed by Western blotting.