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Past Issue in 2013
Volume: 3, Issue: 24
Cancer Biology
Invadopodia Detection and Gelatin Degradation Assay
This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.
Measurement of Acetylcholine from Cell Lines
Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.
Cell Biology
Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)
Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three methods are frequently used to provide basic information about autophagy, people should keep in mind that they are not enough to give the exact details about the autophagy flux due to the complexity of this dynamic process. In addition, acridine orange assay is only a supplementary method to detect autophagy because it also has high affinity to other organelles such as lysosomes. For further investigation about a compound’s effect on autophagy flux, additional and more complicated assays are recommended. Protocols here provide a starting point for people to get a snapshot of whether a compound can affect autophagy in tissue culture cells.
ImmunoFISH for Adherent Cultured Mammalian Cells
This protocol is optimized for immunoFISH staining of adherent cultured mammalian cells. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) and FISH against telomeric DNA.
ImmunoFISH for Mice and Baboons Frozen Sections
This protocol is optimized for immunoFISH staining of OCT section of mouse tissues. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) (Le et al., 2010) and FISH against telomeric DNA.
Immunology
Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction
Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze the molecular interaction, enabling rapid and repetitive estimation with relatively small amount of sample. Here we describe a general protocol about SPR analysis on the interaction between viral antigen and human MAb (HuMAb) IgG. Anti-human Fcγ is first covalently crosslinked on the sensor chip by amine coupling, and then HuMAb of interest is immobilized via anti-Fcγ MAb IgG interaction as ligand. Antigen injected on the sensor chip causes the SPR change in time course as the result of association and dissociation. By analyzing the kinetics, association rate, dissociation rate, and dissociation constant are obtained.
Influenza Virus-cell Fusion Inhibition Assay
During viral infection to host cells, several viruses undergo the process of endocytosis and pH-dependent fusion. By fusion of viral membrane with host cellular membrane, the viral core invades to host cytoplasm. A part of monoclonal antibodies against viral membrane protein have potential to inhibit the viral fusion step. Here we describe in vitro influenza virus-cell fusion inhibition assay. The infected cells expressing viral membrane protein, such as hemagglutinin (HA), on cellular surface are incubated with monoclonal antibodies targeting viral membrane protein. Then the cells are incubated under low pH condition. If the antibody does not inhibit the fusion step, we can see multinucleated giant cells.
Isolation of Neutralizing Antibody
Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. Then each of prepared HuMAbs is confirmed whether it can neutralize the specific virus by in vitro neutralization assay.
Microbiology
EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes
Clostridium difficile is a Gram-positive, spore-forming, strict anaerobe and the leading cause of antibiotic-associated diarrhea (McFarland, 2008). Germination by C. difficile spores is the first step in pathogenesis. Thus, identifying mechanisms of C. difficile spore germination may lead to novel anti-germination therapies. This protocol describes a method for identifying germination-null phenotypes for C. difficile spores by introducing random mutations into actively growing C. difficile using ethyl methanesulfonate (EMS) as a mutagen (Francis et al., 2013).
Colony Forming Assay for HCV-Replicon Cell Line
Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Since the HCV genome is present exclusively in RNA form during replication, a number of anti-HCV drugs show appearance of rapid drug-resistant viruses. Therefore, it is important to test generation of drug-escape mutant viruses by developed antiviral drugs for their validity. Here, we describe a colony formation assay-based method to observe appearance of drug-resistant viruses against nucleic acid based anti-HCV drugs in genotype 1b based subgenomic replicon cell culture system (Lee et al., 2013).
Virulence Studies of Clostridium difficile
Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) can colonize antibiotic-treated hosts (McFarland, 2008). Antibiotics alter the composition of the normal, benign microbial flora which leads to loss of colonization resistance (Wilson and Perini, 1988; Antonopoulos et al., 2009). C. difficile spores germinate to actively growing bacteria which secrete toxins that damage the colonic epithelium (Voth and Ballard, 2005). The use of animal models of C. difficile disease have allowed the identification of mechanisms of colonization and virulence factors (Lyras et al., 2009; Kuehne et al., 2010; Francis et al., 2013; Aubry et al., 2012; Carter et al., 2011). This protocol describes virulence studies of C. difficile in the hamster model of C. difficile infection (Bartlett et al., 1978; Sambol et al., 2001).
Neuroscience
Ex utero Electroporation into Mouse Embryonic Neocortex
This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse Embryo Brains” (Ge, 2012); “Organotypic Slice Culture of Embryonic Brain Sections” (Calderon de Anda, 2013). Additionally, ex utero electroporated neocortices can be used for in vitro primary cell cultures with further dissection, dissociation into single cells, and plating on cover slips or in multi-well dishes according to standard techniques: note this procedure can be performed immediately after electroporation, prior to the onset of ectopic gene expression, or after overnight slice culturing to collect just the region of electroporated cells.
Plant Science
Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings
Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including plasma membrane, intracellular vesicles, Golgi membranes, endoplasma reticulum, and tonoplast (vacuolar membrane) from 5-7 days old seedlings, which is often analyzed for auxin transporters in Arabidopsis (Leonard and Vanderwoude, 1976; Titapiwatanakun, et al., 2009; Yang et al., 2013; Blakeslee et al., 2007). After homogenization, plant debris including cell walls, chloroplasts and nucleus were removed by low speed centrifugation (8,000 x g), then total microsomal membranes were pelleted by high speed centrifugation (10,000 x g) and separated from soluble fractions. We secondly describe a method to separate microsomal fractions according to size or density in a sucrose density-gradient system by centrifugation. The linear sucrose gradient from 20%-55% (1.09-1.26 g cm-3) were used to separate membranes with different densities: tonoplast, 1.10-1.12 cm-3, Golgi membranes, 1.12-1.15 cm-3, rough endoplasmic reticulum 1.15-1.17 cm-3, thylakoids, 1.16-1.18 cm-3, plasma membrane, 1.14-1.17 g cm-3, and mitochondrial membranes, 1.18-1.20 cm-3 (Leonard and Vanderwoude, 1976; Larsson et al., 1987; Briskin and Leonard, 1980). However, the plasma membrane can also be isolated according to its outer surface properties which are very different from intracellular membrane surfaces. Thus, the right-side-out plasma membrane vesicles can be separated in an aqueous Dextran-polyethylene glycol two-phase system. The plasma membranes can be purified to > 90% in the upper phase (Larsson et al., 1987; Alexandersson et al., 2008). Two-phase systems for Arabidopsis seedlings were described in the section 3. Sucrose density gradient membrane fractionation followed by western blot is often used to analyze the distribution of certain membrane protein, while Two-phase separation is used when high purity of plasma membrane or intracellular membrane is required.
Library Construction for Genome-wide Bisulfite Sequencing in Plants
DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis
Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, so that this protocol is useful for analysis of cp-actin dynamics that are induced by blue light.
Stem Cell
Preparation of Adult Mouse Muscle Stem Cells
Muscle stem cells are adult stem cells responsible for muscle development, growth and regeneration. Current knowledge suggests those cells are heterogeneous population shared their position between the sarcolemma and basal lamina of muscle fibers. This protocol describes the technique to dissociate and collect the stem cells from skeletal muscle of adult mice, and separate them from other cells found in muscle (e.g. fat, connective tissue). To purify and preserve those myofiber associated muscle stem cells, we use two steps of enzyme digestion followed by cell pre-plating procedures.
Intestinal Differentiation of Mouse ESCs
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from mouse ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from mouse ESCs.
Intestinal Differentiation of Human ESCs
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from human ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from human ESCs. The protocol for human ESCs is principally very similar with that for the mouse ESCs, with some modifications.