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Past Issue in 2014
Volume: 4, Issue: 22
Cancer Biology
Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC)
Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAM) and tumor-associated dendritic cells (TADC). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Laoui et al., 2011). These cells are able to promote tumor progression via several mechanisms, including induction of angiogenesis, remodeling of the extracellular matrix, stimulation of cancer cell proliferation and metastasis and the inhibition of adaptive immunity (Laoui et al., 2011). Moreover, mononuclear myeloid cells are characterized by plasticity and versatility in response to microenvironmental signals, resulting in different activation states, as illustrated by the presence of distinct functional TAM subsets in tumors (Movahedi et al., 2010; Laoui et al., 2014). Here, we describe a valuable isolation technique for TAM and TADC permitting their molecular and functional characterization.
Induction of Colitis and Colitis-associated Colorectal Cancer (CAC)
Inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis are characterized by chronic, progressive and relapsing inflammatory disorders. Existing evidence indicate that IBD is associated with a higher risk of developing CAC, which is directly related to the duration and extent of colitis. Thus, animal models have been developed to understand the biology of colitis and CAC. The most commonly used model of colitis is to treat with dextran sodium sulfate (DSS). DSS given in the drinking water is toxic to the colonic epithelial lining and induces bloody diarrhea, ulceration and inflammation, similar to colitis in IBD patients. To study CAC, DSS treatment is combined with a single intraperitoneal injection of the DNA alkylation reagent Azoxymethane (AOM).
Cell Biology
Analysis of Intestinal Permeability in Mice
The intestinal epithelial layer serves as a barrier against pathogens and ingested toxins, which are present in the lumen of the intestine. The importance of the intestinal epithelial barrier is emphasized by the alterations in paracellular permeability and tight junction functions observed in inflammatory bowel disease (IBD) and colon cancer.
Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells
Mitochondria are organelles that have important functions in oxidative phosphorylation, fatty acid oxidation and apoptosis signaling. They have two distinct membranes, outer membrane (OM) and inner membrane (IM). IM contains respiratory chain complexes that produce ATP. IM is rich in cardiolipin, a specific phospholipid reportedly having a critical role for organizing super-complex formation of respiratory chain complexes. IM abundant in cardiolipin exhibits resistance to extraction by digitonin (a non-ionic detergent), whereas the detergent easily lyses OM. Therefore, digitonin is useful to separate mitoplast (IM plus matrix) and OM from mitochondria. Here, we describe a method to isolate mitochondria from HeLa cells, and a method to isolate mitochondrial outer membrane proteins and inner membrane proteins by using digitonin. This method is applicable also to other types of cultured cells such as COS-7.
Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities
The polyamines, spermidine (Spd) and spermine, and their diamine precursor putrescine, are important regulators of various cellular functions, such as proliferation and differentiation. Polyamine homeostasis is tightly regulated on the level of uptake, excretion, biosynthesis, interconversion and terminal catabolism. The rate-controlling enzymes of polyamine biosynthesis and interconversion are ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), respectively. Here, we describe a protocol to assay ODC (Jänne and Williams-Ashman, 1971) and SSAT (Libby, 1978) activities from cell or tissue samples.
Preparation of Golgi Membranes from Rat Liver
This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from Leelavathi et al. (1970).
Microbiology
Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses
Titration of viral stocks is a critical process before any experimental use of the virus. Here we describe an infectious focus assay for several alphaherpesviruses, a titration method for fluorescently labeled viruses, based on the original plaque assay. In addition, the calculation of multiplicity of infection (MOI) is presented.
Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also have beneficial adaptive roles in lower eukaryotes (in fungi and yeast) (Eaglestone et al., 1999; True et al., 2004; Coustou et al., 1999). Besides separating polymers from their precursor soluble monomers, another particular difficulty of the study of amyloid proteins is to resolve the heterogeneity of the aggregates, since these usually exhibit a variable degree of polymorphism. Semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) is a technique that takes advantage of both the property of prions and prion-like polymers to be highly resistant to solubilization by SDS detergent, and the large pores sizes of agarose, that allow the resolution of high molecular weight complexes. In this method, we describe in detail how this technique can be used to characterize heterogeneous aggregation in bacteria and yeast (Gasset-Rosa et al., 2014; Molina-García and Giraldo, 2014), and further be applied to study the aggregation pattern of proteins that become prone to aggregation through genetic manipulation.
Isolation and Characterisation of Dendritic Cells from Peripheral Blood
Latency and reactivation of human cytomegalovirus (HCMV) is intimately associated with the myeloid lineage. Multiple studies have used in vitro protocols to generate dendritic cells (DCs) from myeloid precursors. Here we describe the direct isolation of DCs from peripheral blood to study HCMV latency directly in this cell type.
Competitive ELISA for Protein-lipopolysaccharide (LPS) Binding
Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pIet al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006). Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.
Purification and Sequencing of DNA Guides from Prokaryotic Argonaute
Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand matches the ssDNA molecule is not lost during this process.
Secretion Assay in Shigella flexneri
Shigella flexneri (S. flexneri) is a Gram-negative bacterium that causes gastroenteritis and shigellosis in humans. In order to establish and maintain an infection, S. flexneri utilises a type three secretion system (T3SS) to deliver virulence factors called effector proteins into the cytoplasm of host cells, facilitating e.g. uptake into the host cell and escape from the endosome. Secretion through the T3SS is tightly regulated and is usually triggered by host-cell contact, but can also be artificially stimulated in vitro. In this assay, the dye Congo red is used to induce T3SS-dependent secretion of S. flexneri (Parsot et al., 1995) and secreted proteins are concentrated from the culture supernatant by precipitation with trichloroacetic acid. The assay presented here can easily be adapted to the secretion analysis of other bacteria utilising a T3SS, such as Salmonella typhimurium, which constitutively secrete when grown at 37 °C (Collazo et al., 1995; Pegues et al., 1995), or pathogenic species of Yersinia, where secretion can be induced by calcium deprivation (Heesemann et al., 1986; Forsberg et al., 1987).
Plant Science
In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique
Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described an easy protocol for quick in vitro detection of protein S-acylation using the Arabidopsis protein kinase, PBS1, as an example. To determine whether PBS1 is modified through thioester linkage to acyl groups, we employed a “biotin switch” assay (Hemsley et al., 2008). This work was first published in Qi et al. (2014), but we expand on the method here. PBS1 functions within the basal immune system of plants, and is a target of the bacterial cysteine protease, AvrPphB (Shao et al., 2002; Zhang et al., 2010). It contains a predicted N-terminal S-acylation motif (MGCFSCFDS), with both Cys-3 and Cys-6 residues predicted to be palmitoylated by CSS-Palm 3.0 (http://csspalm.biocuckoo.org/; Ren et al., 2008). Our method utilizes hydroxylamine-induced cleavage of thioester bonds, which results in free sulfhydryl groups that can then be conjugated to a biotin derivative, 1-biotinamido-4-[4′-(maleimidomethyl) cyclohexanecarboxamido]-butane (Biotin-BMCC). The conjugates are detectable by Western blot with streptavidin-horseradish peroxidase. The whole process of in vitro labelling and detection took less than 3 days, allowing the fast detection of protein modifications via thioester bonds such as palmitoylation.
Stem Cell
Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting
A population of muscle resident CD45-, CD31- cells expressing the mesenchymal PDGF receptor alpha (PDGFRα) as well as Sca-1 was first isolated in healthy mouse muscles in Uezumi et al. (2010). In the same year, Joe et al. (2010) identified and purified fibro-adipogenic precursors (FAPs), cells located into the interstitial space between myofibers close to vessels, negative for CD45, CD31,α7-Integrin, but expressing CD34, Sca-1. Both groups demonstrated that these cells are not myogenic in vitro or in vivo, but they are capable of differentiating in vitro towards both fibrogenic and adipogenic lineage (Uezumi et al., 2011). Further marker analysis indicates that the two groups identified independently the same cell population (Natarajan et al., 2010).FAPs are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle (Natarajan et al., 2010; Cordani et al., 2014). We have recently demonstrated that Nitric Oxide regulates FAP fate inhibiting in vitro their differentiation into adipocytes. In mdx mice, an animal model of DMD, fed with a diet containing the nitric oxide donating drug, Molsidomine, the number of PDGFRα+ cells was reduced as well as the deposition of both skeletal muscle fat and connective tissues (Cordani et al., 2014). Here we described a method to isolate in both wild type and in mdx dystrophic muscle pure population of FAPs by double selection for SCA-1 and PDGFRα positivity in absence of the satellite cell markers SM/C2.6 and α7integrin as well of the pan-lymphocytes marker CD45 or endothelial marker CD31.