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Past Issue in 2014
Volume: 4, Issue: 14
Cancer Biology
Bone Resorption Assay
The Bone resorption assay provides an easy to use protocol for quantitatively measuring in vitro osteoclast-mediated bone resorption. Osteoclasts can be seeded onto the bone slices and formation of resorption pits can be quantified via toluidinblue staining (Scholtysek et al., 2013).
Developmental Biology
Wnt Reporter Activity Assay
This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.
Hedgehog (Hh) Reporter Activity Assay
This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.
Fluorescence Microscopy for Cilia in Cultured Cells and Zebrafish Embryos
Cilia are microtubule-based hair-like projections found in organisms, ranging from protozoa to mammals. This protocol provides methods for immunofluorescence staining of cilia in cultured cells and zebrafish embryos.
Immunology
Macrophage Inflammatory Assay
Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’.In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16-18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).
Isolation of ILC2 from Mouse Liver
Group 2 innate lymphoid cells (ILC2) are a recently characterized cell population which lacks specific antigen receptors and contributes to immune responses at mucosal surfaces of lung and gut. Recently, we demonstrated that ILC2 expand in the context of chronic liver diseases in mice and contribute to pathology in an IL-33 dependent manner. Here, we describe a protocol to isolate highly purified ILC2 from mouse livers. This procedure provides effective digestion of liver tissue and limits proteolytic degradation of cell surface receptors leading to increased yields of biologically functional cells. The number of liver resident ILC2 in the steady state is low, however their number dramatically increase upon systemic or local treatment of mice with cytokines such as IL-25 and IL-33. Using minicircle-based expression constructs for these cytokines high numbers of functional ILC2 can be isolated with the protocol provided here.
Microbiology
Chromatin Fractionation Assay in Fission Yeast
The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us to fractionate chromatin-bound and -unbound proteins from living cells. The following immunoblot of the respective fractions provides the information concerning the chromatin binding status of our target proteins.
Dye Release Experiments with Dextran Loaded Vesicles
Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013).
Development of a Novel Assay for Synthesis and Hydrolysis of Sedoheptulose 1,7-bisphosphate (SBP) in vitro by Combinations of Purified Fructose 1,6-bisphosphate aldolases (FBA) Proteins and Fructose 1,6-bisphosphatases (FBPase) Proteins from Bacillus methanolicus MGA3
Bacillus methanolicus (B. methanolicus) is a Gram-positive, thermotolerant, and facultative methylotrophic bacterium that can use the one-carbon (C1) compound methanol as a source of carbon and energy (Schendel et al., 1990; Arfman et al., 1997; Arfman et al., 1992). B. methanolicus uses the Ribulose monosphosphate (RuMP) cycle for the fixation of formaldehyde (Anthony, 1986; Brautaset et al., 2007). In the RuMP cycle, Sedoheptulose 1,7-bisphoasphate (SBP) can be produced from erythrose 4-phosohate (E4-P) and dihydroxyacetone phosphate (DHAP) by sedoheptulose 1,7-bisphosphate aldolase (SBA) and dephosphorylated to yield sedoheptulose 7-phosphate (S7-P) by sedoheptulose 1,7-bisphosphatase (SBPase). Unfortunately, since neither E4-P nor SBP is commercially available, these compounds cannot be used directly in enzyme assays to obtain evidence for synthesis and hydrolysis of SBP. To circumvent this limitation, a coupled discontinuous enzyme assay including transketolase from Saccharomyces cerevisiae (S. cerevisiae) was used. E4-P and xylulose 5-phosphate (XU5-P) were generated from fructose 6-phosphate (F-6P) and glyceraldehyde 3-phosphate (GAP) by transketolase from S. cerevisiae. Aldol condensation of E-4P with DHAP to yield SBP was tested by using purified fructose 1,6-bisphosphate aldolase (FBAC or FBAP) from B. methanolicus (Stolzenberger et al., 2013a). Subsequently, hydrolysis of SBP to S7-P was assayed by using purified fructose 1,6-bisphosphatase (GlpXC or GlpXP) from B. methanolicus (Aldolases and phosphatases are not commercially available.) (Stolzenberger et al., 2013b).
Bacterial Fluorescent-dextran Diffusion Assay
Antimicrobial peptides are known to disrupt bacterial membranes allowing solutes to flow across the membrane in an unregulated manner resulting in death of the organism. Disrupting the bacterial membrane would thus perturb the cells osmotic balance resulting in an initial influx of the external aqueous buffer. We have designed an assay to investigate how antimicrobial peptide concentration affects the ability of fluorescently labelled dextran moieties of differing molecular weight and hydrodynamic radii to cross membranes of viable bacteria. This assay was used to show that diffusion of low and high molecular weight dextrans into bacteria was a function of antimicrobial peptide concentration (Sani et al., 2013).
Affinofile Assay for Identifying Macrophage-Tropic HIV-1
The ability to enter monocyte-derived macrophage (MDM) in vitro is commonly used to define macrophage-tropic HIV-1 despite the fact that viruses vary continuously in their ability to enter MDMs in vitro, and MDMs vary in their ability to support HIV-1 entry (Joseph et al., 2014; Peters et al., 2006). This makes it difficult to distinguish viruses that are adapted to replicating in macrophage from those that are adapted to replicating in T cells. We use the Affinofile cell line ( Johnston et al., 2009) to assay for macrophage tropism by capitalizing on the fact that macrophage-tropic HIV-1 has an enhanced ability to enter cells expressing low levels of CD4 (Joseph et al., 2014; Peters et al., 2006; Duenas-Decamp et al., 2009; Dunfee et al., 2006; Gorry et al., 2002; Martin-Garcia et al., 2006; Peters et al., 2004) and Affinofile cells can be induced to express a wide range of CD4 densities (Johnston et al., 2009). We induce Affinofile cells to express either high or low CD4, infect those cells with pseudotyped reporter virus, and quantify percent infectivity at low CD4 relative to infectivity at high CD4. Macrophage-tropic viruses have an enhanced ability to infect at low CD4. Using this approach we have found that macrophage-tropic strains of HIV-1 are relatively rare and that most HIV-1 variants require high levels of CD4 to enter cells, a phenotype we have referred to as R5 T cell-tropic.
In vitro EBV Infection of Mononuclear Cells that Have Been Cryo-preserved
Epstein-Barr Virus (EBV) is a B-lymphotropic herpesvirus which the majority of adult human population is latently-infected with. Various immunological and molecular in vitro studies have been facilitated by the use of EBV’s ability to infect and transform B cells to immortalized polyclonal B cell lines. Many of these studies use freshly isolated cord-blood mononuclear cells (CBMC). Some experiments may, however, require EBV infection of samples that have been prospectively collected and cryo-preserved. Here we share a protocol that we used to successfully infect B cells from cryo-preserved CBMCs and peripheral-blood mononuclear cells (PBMC) (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013).
Plant Science
Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells
In this protocol, we describe a simple and efficient method for meiotic chromosome spread preparation in rice pollen mother cells. Meiotic chromosome preparation by spreading itself is an important technique for plant cytogenetics (Higgins et al., 2004; Chelysheva et al., 2012; Wang et al., 2009); furthermore, it is a crucial step for applying other cytogenetic methods including Fluorescence in situ hybridization (FISH) and immunostaining.
Stem Cell
Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo
Human mesenchymal stem/progenitor cells (MSCs) isolated from various adult tissues show remarkable therapeutic potential and are being employed in clinical trials for the treatment of numerous diseases (Prockop et al., 2010). While routes of cell administration vary, profound beneficial effects of MSCs in animal models have been observed following intraperitoneal injections of the cells (Roddy et al., 2011). Similar to MSC spheres formed in culture under conditions where attachment to plastic is not permitted (Bartosh et al., 2010), MSCs injected into the peritoneum of mice spontaneously aggregate into 3D sphere-like structures (Bartosh et al., 2013). During the process of sphere assembly and compaction, MSCs upregulate expression of numerous therapeutic anti-inflammatory and immune modulatory factors. Here we describe the method we previously used for the generation of human bone marrow-derived MSC aggregates/spheres in vivo (Bartosh et al., 2013). By tagging the MSCs with green fluorescent protein (GFP), the aggregates formed can be easily visualized, collected and analyzed for changes in cellular properties and interactions with host immune cells.
Coculture between hMADS and Mouse Adult CM
Heart failure occurring after acute myocardial infarction (MI) is among the main causes of death in western countries. Cell therapies, particularly those based on mesenchymal stem cells (MSC), represent one of the most promising approaches to repair damaged heart tissues. Several reports have provided evidences that injection of mesenchymal stem cells improved heart function following myocardial infarction (Shake et al., 2002; Zimmet and Hare, 2005; Zeng et al., 2007). Nevertheless, the mechanism(s) by which MSC exert their therapeutic action is far from being understood, and further knowledges in this field are required especially to optimize efficiency of current cardiac cell therapies. To assess the regenerative mechanisms developed by MSC in vitro, we developed the method described above which is expected to mimic the micro-environment typical of an infarcted heart. This method consists in a species mismatch coculture between mouse terminally differentiated cardiomyocytes in a distressed state and human Multipotent Adipose Derived Stem cells (hMADS cells) used herein as an MSC model.