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Past Issue in 2015
Volume: 5, Issue: 20
Cancer Biology
Rat Aortic Ring Model to Assay Angiogenesis ex vivo
Angiogenesis is a multifactorial event which requires the migration, proliferation, differentiation and structure rearrangement of endothelial cells. This angiogenic process has been commonly studied using in vitro assays such as Boyden chamber assay, wound healing assay and tube formation assay. These assays mainly use monolayers of endothelial cells which are modified by repeated passages and are fully proliferative, a situation far away from physiology. In addition, not only endothelial cells are involved in this process but surrounding cells (such as pericytes, smooth muscle cells, fibroblasts) and the supporting matrix are also major players. The three-dimensional ex vivo aortic ring model recapitulates the complexities of angiogenesis and combines the advantages of in vitro and in vivo models. The aortic ring is cultivated in a chemically defined culture environment. Microvessels which grow in this system are lumenized vessels with surrounding supporting cells and are essentially indistinguishable from microvessels formed during angiogenesis in vivo. The efficacy of pro-or anti-angiogenic factors can be determined in the absence of serum molecules which may otherwise interfere with the substances being tested (Nicosia and Ottinetti, 1990). However, this system requires access to fresh rat tissue but several samples can be prepared from one aorta.
Cell Biology
In vivo Fluorescein Isothiocyanate-dextran (FD4) Permeability Assay
Using pluripotent stem cells, it is now becoming possible to develop tissue models of organ systems within the body. These organs will allow for the study of organ function, physiology, embryology, and even pathologic processes. Recently, our group developed a model of human small intestine developed from human pluripotent stem cells which when transplanted in vivo, produce a mature, cystic intestinal structure that has digestive functions similar to that of native small intestine (Watson et al., 2014). Intestinal permeability is a primordial function of both the epithelium and associated tight junctions to control nutrient intake and prevent the passage of pathogens. One way to study gastrointestinal paracellular permeability is by determining the ability of fluorophores-conjugated macromolecules (i.e., fluorescein isothiocyanate-dextran (FITC-dextran; or FD4) to cross from the lumen and into circulation (Dong et al., 2014). We were able to test the intestinal permeability by injecting FITC-dextran directly into the lumen of the bioengineered intestine and determining the fluorescence within the blood of the murine host at various time points after injection.
Intravenous Tomato Lectin Injection to Assess Functional Vasculature
Pluripotent stem cells have recently allowed for the development of tissue models for the various organ systems within the body. These models allow scientists to study organ function, physiology, embryology, and even pathologic processes. Studies on tissue can be done in vitro and/or transplanted into animal models for studies in vivo. Recently, our lab developed a model of human small intestine derived from human pluripotent stem cells which when transplanted in vivo, matured into an intestinal structure similar to that of adult intestine. The maturity of the transplanted human intestinal tissue was dependent upon the development of an adequate blood supply primarily from the murine host. In order to better study the developed vascular network within our transplanted intestinal tissue, we injected Fluorescein labeled Lycopersicon esculentum (tomato) lectin into the mouse tail vein (Watson et al., 2014). Using the property of this lectin to bind to the endothelium, we were able to visualize the vasculature within the transplant.
Immunology
Immune Cell Isolation from Mouse Femur Bone Marrow
The bone marrow is the site of hematopoiesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.
Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts
Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency.
Collagen-induced Arthritis: A Model for Murine Autoimmune Arthritis
Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days after the first injection. These mice typically develop disease 26 to 35 days after the initial injection. C57BL/6J mice are resistant to arthritis induced by type II bovine collagen, but can develop arthritis when immunized with type II chicken collagen in CFA, and receive a boost of type II chicken collagen in IFA 21 days after the first injection. The concentration of heat-killed Mycobacterium tuberculosis H37RA (MT) in CFA also differs for each strain. DBA/1J mice develop arthritis with 1 mg/ml MT, while C57BL/6J mice require and 3-4 mg/ml MT in order to develop arthritis. CIA develops slowly in C57BL/6J mice and cases of arthritis are mild when compared to DBA/1J mice. This protocol describes immunization of DBA/1J mice with type II bovine collagen and the immunization of C57BL/6J mice with type II chicken collagen.
Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays
Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope-specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused on developing an HIV-1 vaccine, we immunized rabbits with hyperglycosylated stable core immunogens, which were designed using high-resolution structural information to elicit antibodies against the primary receptor-binding, CD4-binding site on HIV-1 gp120. Using a solid phase adsorption assay, we could map the serum antibodies to the conserved CD4-binding site, a known broadly neutralizing determinant on exterior envelope glycoprotein, gp120.
Microbiology
Product Analysis of Starch Active Enzymes by TLC
Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).
Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods for purification of this protein from L. otakiensis JCM 15040. Because no specific ligand that has high affinity for this enzyme has been identified, the purification was performed using ammonium sulfate fraction, four types of column chromatography and preparative Native-PAGE, not using an affinity column chromatography. We hope that the protocol will provide useful information for purification of an enzyme that cannot easily be purified using an affinity column chromatography.
Plant Science
Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis
Hyaloperonospora arabidopsidis (Hpa; formerly Peronospora parasitica or Hyaloperonospora parasitica) is an oomycete downy mildew pathogen of the model plant Arabidopsis. The pathosystem between Arabidopsis and Hpa has been extensively used to study host/pathogen co-evolution (Coates and Beynon, 2010). As Hpa is an obligate biotrophic pathogen, its host is absolutely required for survival. Thus, Hpa must be maintained on susceptible Arabidopsis accessions and mutants. Growth of Hpa is evaluated in two ways; counting conidiospores (Asai et al., 2014) or counting sporangiophores after trypan blue staining (Holt et al., 2005). Here, we describe how to do inoculation with Hpa and how to evaluate Hpa growth on Arabidopsis.
Isolation of Triterpenes from Propolis (Bee Glue)
Propolis (bee glue) is a natural substance produced by bees upon collection of mainly plant resins. Bees use it as antiseptic sealing agent between honeycombs and to preserve the hive from external contamination. Numerous scientific studies have been published on the biological properties of propolis including its anti-inflammatory, anti-oxidant, immunostimulant, antitumour and antimicrobial activity. Different propolis chemotypes have been characterised based on the nature of the plant-derived substances present and the geographical origin of collection. Here, we describe the isolation of nine triterpenes from a sample of propolis originating from North-Western Cameroon. All compounds were identified following analysis of their spectroscopic data and comparison with previously published reports.
Isolation and Characterization Procedure for Indole Alkaloids from the Marquesan Plant Rauvolfia Nukuhivensis
A plethora of natural products, mostly secondary metabolites, are isolated and purified from many different organisms, like plants, fungi, algae, marine invertebrates, etc. The extraction procedure is specific to each organism, but some guidelines are usually followed for any purification procedure regarding targeted metabolites, such as alkaloids. Alkaloids are secondary metabolites that contain basic nitrogen in their structures and they are often associated with interesting biological properties especially in pharmacology field. This protocol describes the isolation procedure of indole alkaloids from Rauvolfia nukuhivensis directly from the ethanol extract of the plant material yielding different skeleton-type compounds including non-basic derivatives (ajmaline, sarpagine, macroline and β-carboline). The procedure details the guidelines and the steps to characterize new or known isolated compounds, beginning from the plant collection to the molecule level with the use of spectroscopic techniques (NMR, MS, UV). We detailed the extraction and fractionation procedures followed by the purification of compounds, as well as their physico-chemical characterizations. The procedure is illustrated by the example of the purification of a large array of indole alkaloids from the bark of Rauvolfia nukuhivensis.
Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues
This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.
Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells
Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al., 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.
Vacuole Structure Analysis during Cell Death Subsequent to Application of Erwinia carotovora Culture Filtrates to Cell Cultures of Nicotiana tabacum
We recently established an experimental model system for efficient defense-related cell death using tobacco BY-2 cultured cells treated with culture filtrates of the pathogenic bacterium Erwinia carotovora (E. carotovora) (Hirakawa et al., 2015). Applying this experimental system to transgenic BY-2 cells stably expressing the vacuolar membrane marker GFP-VAM3 (Kutsuna and Hasezawa, 2002) allowed us to monitor changes in vacuolar membrane structures including a decrease of transvacuolar strands during cell death (Hirakawa et al., 2015). Our model system can help to investigate organelle dynamics in defense-related cell death. Here, we show protocol for applying E. carotovora filtrates to BY-2 cells and confocal observation of vacuolar membrane dynamics and subsequent cell death. We used cell cycle synchronized BY-2 cells to effectively monitor invaginated vacuolar membranes such as transvacuolar strands in our recent report (Hirakawa et al., 2015); however, we do not describe the protocol for cell cycle synchronization in this article. For the step-by-step protocol for BY-2 cell synchronization, please refer to previous protocol papers (Nagata and Kumagai, 1999; Kumagai-Sano et al., 2006).
Stem Cell
Clonal Culture of Mouse Liver Progenitor Cells
Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. For analyzing their differentiation potential, clonal culture has been used for LPCs isolated by a cell sorter. In addition, we can use the culture to assess functions of target genes on differentiation potential of LPCs. This protocol describes the process of cell isolation and colony assay to examine proliferative and differentiation potential of LPCs.