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Past Issue in 2015
Volume: 5, Issue: 21
Cancer Biology
Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells
Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combing both targeted- and immuno-therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying the effects of the macrophage secretome on cancer cells (on e.g., growth, drug response or gene expression) in co-cultures without direct cell contact interactions. This is an important aspect as it removes the presence of any phagocytic aspect to changes in the cancer cell number and behaviour. The in vitro THP-1 monocyte differentiation into polarized macrophages was used to study the effects of both M1 and M2 type populations of macrophages on melanoma cells (Smith et al., 2014; Tsuchiya et al., 1980). M1 type macrophages are classically thought to be tumour suppressing as opposed to M2 type macrophages, which are thought to possess tissue repairing and tumour growth promoting activities.
Isolation and Flow-cytometric Analysis of Mouse Intestinal Crypt Cells
The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).
Capturing the Driving Role of Tumor-host Crosstalk in a Dynamical Model of Tumor Growth
In 1999, Hahnfeldt et al. proposed a mathematical model for tumor growth as dictated by reciprocal communications between tumor and its associated vasculature, introducing the idea that a tumor is supported by a dynamic, rather than a static, carrying capacity. In this original paper, the carrying capacity was equated with the variable tumor vascular support resulting from the net effect of tumor-derived angiogenesis stimulators and inhibitors. This dynamic carrying capacity model was further abstracted and developed in our recent publication to depict the more general situation where there is an interaction between the tumor and its supportive host tissue; in that case, as a function of host aging (Benzekry et al., 2014). This allowed us to predict a range of host changes that may be occurring with age that impact tumor dynamics. More generally, the basic formalism described here can be (and has been), extended to the therapeutic context using additional optimization criteria (Hahnfeldt et al., 1999). The model depends on three parameters: One for the tumor cell proliferation kinetics, one for the stimulation of the stromal support, and one for its inhibition, as well as two initial conditions. We describe here the numerical method to estimate these parameters from longitudinal tumor volume measurements.
Cell Biology
Culture of Megakaryocytes from Human Peripheral Blood Mononuclear Cells
Megakaryocytes are the precursor cells of platelets and are bona fide resident cells in the bone marrow but extremely low in numbers (~1% of total nucleated cells). Upon terminal differentiation, megakaryocytes increase their size, become polyploid and develop a demarcation membrane system. Mature megakaryocytes form proplatelets, which are cytoplasmic extensions that protrude through the endothelial cell layer of venous sinusoids within the bone marrow, entering into the blood circulation and, subsequently, releasing platelets. Despite limited in numbers, megakaryocytes have been successfully isolated from bone marrow (Tolhurst et al., 2012), adult peripheral blood (Mazur et al., 1990; Thornton et al., 1999), cord blood (Sun et al., 2004) and also from embryonic stem cells (Pick et al., 2013; Eto et al., 2002). These procedures rely on immunostaining using antibodies against megakaryocyte surface markers (i.e. CD41 or CD42b) to isolate an enriched population of megakaryocytes. Here, we describe a culture method wherein megakaryocytes can be grown and differentiated in vitro from human peripheral blood mononuclear cells (PBMCs) directly without the need of initial isolation of CD34+ cells. This method is based on a previously published culture method of human erythroid progenitor cells from PBMCs (Borg et al., 2010; Leberbauer et al., 2005). Although the purity of megakaryocytes is not 100% in this culture method, an enriched fraction of megakaryocytes can be further isolated using BSA gradient or cell-sorting techniques. In addition, our method offers the possibility to freeze the cultures after minimal expansion of yet undifferentiated megakaryocytes, which will yield equal megakaryocyte cultures after thawing when compared to fresh uninterrupted cultures. As this has been proven difficult with CD34+ sorted pluripotent cells, it allows managing samples and to perform downstream analysis when human material is not always available.
Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry
Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of norepinephrine and free fatty acids are used to induce uncoupled respiration. The ATP Synthase inhibitor oligomycin, the chemical uncoupler FCCP, and the complex III inhibitor Antimycin A are then used to measure coupled, maximal, and non-mitochondrial oxygen consumption, respectively. In the protocol Part II, the plasma membrane is permeabilized with recombinant perfringolysin O, a cholesterol-dependent cytolysin that oligomerizes into pores exclusively in the plasma membrane. This permits experimental control of metabolite availability without separating mitochondria from the native cell environment.
Microbiology
Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed a two-step cross-linking strategy using DSG and formaldehyde-rather than the one-step formaldehyde cross-linking procedure more frequently employed for analyzing direct protein-DNA interactions - in order to better capture the indirect MoJmjC-MoSOD1 DNA interactions in vivo. In addition, we have shown that two-step cross-linking is suitable for ChIP analysis of direct protein-DNA interactions between a GATA transcription factor, Asd4, and its cognate binding site (Marroquin-Guzman and Wilson, 2015). Here, we provide a detailed protocol for chromatin immunoprecipitation, with versatile two-step cross-linking, in M. oryzae.
Determination of Fructokinase Activity from Zobellia galactanivorans
Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of Zobellia galactanivorans. The ZgFK enzyme catalyzes the conversion of fructose to fructose-6-phosphate using ATP as a cofactor.[Principle] Fructokinase (FK) activity was determined by an enzyme-coupled assay (Figure 1). ADP formed through the FK reaction, i.e., phosphorylation of fructose to fructose-6-phosphate (F6P), is used by the pyruvate kinase (PK) which transforms phosphoenolpyruvate (PEP) to pyruvate. Then, lactate dehydrogenase (LDH) converts pyruvate to lactate using NADH as a cofactor. FK activity is measured by following the decrease in absorbance at 340 nm which corresponds to the transformation of NADH to NAD+.Figure 1. Enzyme-coupled reaction used for determination of fructokinase (FK) activity
Determination of Mannitol-2-dehydrogenase Activity
Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) of Zobellia galactanivorans. The ZgM2DH enzyme catalyzes the reversible conversion of mannitol to fructose using NAD+ as a cofactor. ZgM2DH activity was assayed in both directions, i.e., fructose reduction and mannitol oxidation.Reversible reaction:D-mannitol + NAD+ ↔ D-fructose + NADH + H+
Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site cysteine. Membrane topology of proteins can be predicted using different programs and the actual membrane topology can be determined by monitoring the accessibility of cysteine residues introduced in periplasmic (exposed) or cytoplasmic (not exposed) loops to alkylating agents. A two-step protocol is described where whole Escherichia coli (E. coli) cells are first treated with or without a membrane impermeable thiol reagent (2-sulfonatoethyl)-methane thiosulfonate (MTSES) and subsequently labeled with an alkylating reagent maleimide polyethyleneglycol (malPEG). When cysteine residues are accessible to MTSES, and thus exposed to (or accessible from) the periplasm, their free thiol groups covalently react with MTSES and consequently, are blocked for alkylation with malPEG. The thiol groups of cytoplasmic or membrane-embedded cysteine residues are not accessible to MTSES and proteins can be alkylated with malPEG resulting in an increase in molecular weight of 5 kDa. In the second part of the protocol, accessibility of cysteine residues is used to address the acylation state of enzymes that form stable thioester acyl intermediates. Thioesters can be specifically cleaved by neutral hydroxylamine, leading to a free thiol group of the active site cysteine that can then be alkylated with malPEG.
Neuroscience
Electroretinogram (ERG) Recordings from Drosophila
Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model.The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).
Plant Science
Computational Identification of MicroRNA-targeted Nucleotide-binding Site-leucine-rich Repeat Genes in Plants
Plant genomes harbor dozens to hundreds of nucleotide-binding site-leucine-rich repeat (NBS-LRR, NBS for short) type disease resistance genes (Shao et al., 2014; Zhang et al., 2015). Proper regulation of these genes is important for normal growth of plants by reducing unnecessary fitness costs in the absence of pathogen infection. Recent studies have revealed that microRNAs are involved in regulation of NBS genes in plants (Zhai et al., 2011; Shivaprasad et al., 2012). This protocol describes computational methods for the genome-wide identification of plant NBS genes potentially regulated by microRNAs.
Observation of Chloroplast Movement in Vallisneria
Chloroplasts accumulate to weak light and escape from strong light. These light-induced responses have been known from the 19th century (Böhm, 1856). Up to now, many scientists have developed different methods to investigate these dynamic phenomena in a variety of plant species including the model plant Arabidopsis thaliana, a terrestrial dicot (Wada, 2013). Especially, a serial recording to trace the position of individual chloroplast for the analysis of its mode of movement is critical to understand the underlying mechanism. An aquatic monocot Vallisneria (Alismatales: Hydrocharitaceae, Figure 1A) has contributed over a century to such investigation (Senn, 1908; Zurzycki, 1955; Seitz, 1967), because Vallisneria leaves have rectangular parallelepiped-shaped epidermal cells aligned orderly in a monolayer (Figure 1B), providing an excellent experimental system for microscopic studies. Here we describe a protocol for the up-to-date time-lapse imaging procedures to analyze Vallisneria chloroplast movement. Using this and prototype procedures, the relevant photoreceptor systems (Izutani et al., 1990; Dong et al., 1995; Sakai et al., 2015), association with actin cytoskeleton (Dong et al., 1996; Dong et al., 1998; Sakai and Takagi 2005; Sakurai et al., 2005), and regulatory roles of Ca2+ (Sakai et al., 2015) have been strenuously investigated.Figure 1. Vallisneria plant. A. Whole plant body; B. A bright-field image of adaxial epidermal cells containing a large number of chloroplasts; C. Culture facilities.
In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified first as the photoreceptor for hypocotyl phototropism in Arabidopsis thaliana, and now known to regulate many responses including chloroplast photorelocation movements in various plant species (Christie, 2007). Phototropins are localized mainly on the plasma membrane and their auto-phosphorylation induced by blue light is the critical step of signal transduction pathway (Sakamoto and Briggs, 2002; Kong et al., 2006; Kong et al., 2013; Inoue et al., 2010). Here we describe a protocol for in vitro protein phosphorylation assay using crude-microsomal and plasma-membrane-enriched fractions of Vallisneria, which enabled us to verify the presence of phototropins and characterize their auto-phosphorylation responses. After these analyses, Sakai et al. (2015) proposed that Vallisneria phototropins mediate the high-intensity-blue-light-induced chloroplast avoidance response.
Stem Cell
Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro
Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.
Skeletal Myogenesis in vitro
Mature skeletal myofibers are elongated and multinucleated cells. Many stem/progenitor cell types, including committed muscle stem (satellite cells) and progenitor (myoblasts) cells, muscle-derived stem cells, myogenic endothelial cells, and mesenchymal stem/stromal cells, have been shown to exhibit skeletal myogenesis under appropriate inductive conditions. Committed muscle stem/progenitor cells and multipotent stem/progenitor cells which have skeletal myogenic capacity can typically be differentiated into skeletal myofibers in vitro following extended low-serum exposure. Differentiated cells exhibit distinct fiber-like elongated morphology with multiple nuclei and express unique muscle molecular markers indicating myogenesis, including desmin (early) and fast- and/or slow-myosin heavy chain (mature).