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Past Issue in 2017
Volume: 7, Issue: 17
Cancer Biology
Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.
Protocol for Establishing a Multiplex Image-based Autophagy RNAi Screen in Cell Cultures
Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy is regulated provides an opportunity to explore druggable targets and subsequently develop treatment strategies for these diseases. To identify novel autophagy regulators, we developed an image-based phenotypic RNAi screening approach using autophagic marker proteins at endogenous levels (Jung et al., 2017). In contrast to previously performed autophagy screens, this approach does not use overexpressed, tagged autophagy marker proteins but rather detects autophagic structures at endogenous levels. Furthermore, we monitored early and late phases of autophagy in parallel while other screens employed only a single autophagosome marker mostly GFP-LC3B. Here, we describe this multiplex screening protocol in detail and discuss general considerations about how to establish image-based siRNA screens.
GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).
Cell Biology
Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging
One of the most prominent applications of fluorescent super-resolution microscopy is the study of nanodomain arrangements of receptors and the endocytic pathway. Staining methods are becoming crucial for answering questions on the nanoscale, therefore, the use of small and monovalent affinity probes is of great interest in super-resolution microscopy with biological samples. One kind of affinity probe is the aptamer. Aptamers are single DNA or RNA sequences that bind with high affinity to their targets and due to their small size they are able to (i) place the fluorophore in close proximity to the protein of interest and (ii) bind to most of the protein of interest overcoming the steric hindrance effect, resulting in better staining density. Here we describe a detailed protocol with which to stain live cells using aptamers and to image them with Stimulated Emission Depletion (STED) microscopy. In this protocol, the stainings were performed with commercially available aptamers that target the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2 or ErbB2) and the ephrin type-A receptor 2 (Epha2). Since aptamers can be coupled to most of the popular fluorophores, we believe that the procedure presented here can be extended to the large majority of the current super-resolution microscopy techniques.
Peroxisome Motility Measurement and Quantification Assay
Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.
Developmental Biology
Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).
Immunology
In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay
One-way mixed lymphocyte reaction (MLR) is a classic tool to measure how T cells react to external stimuli. However, MLR is an in vitro reaction system, which shows different response intensity compared with in vivo trails sometimes due to the lack of cytokines, tissue matrix and other immune response associated factors. The following popliteal lymph node assay (PLNA) protocol is designed to test the T cells antigen-specific reaction in vivo by using ovalbumin (OVA) specific reacted transgenic mouse OT-1 and OT-2.
Microbiology
Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)
Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ROS by using an oxidant-sensing probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in cyanobacteria. This method provides reliable, simple, rapid and cost effective means for detection of ROS in cyanobacteria.
Rearing of Culex spp. and Aedes spp. Mosquitoes
Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.
Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is then purified in a buffer containing dodecyl maltoside using Strep-Tactin affinity chromatography. Following enzymatic cleavage of the attached GFP and Strep-tag by thrombin, the P2X7 receptor is isolated using size exclusion chromatography. This method typically yields ~2 mg of purified protein from 6 L of Sf9 culture. Purified protein can be stored in a buffer containing 15% glycerol at 4 °C for at least 2 months and used for a variety of functional and structural studies (Karasawa and Kawate, 2016).
Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells
Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED increases resolution compared to confocal microscopy, which can be further improved by image processing using deconvolution software.
Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R)
The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex (Minamino et al., 2011). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex (Minamino et al., 2016). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have developed an in vivo pH imaging system with high spatial and pH resolution (Morimoto et al., 2016). Here, we describe how we measure the local pH near the export apparatus in living Salmonella cells (Morimoto et al., 2016). Our approach can be applied to a wide range of living cells. Because local pH is one of the most important parameters to monitor cellular activities of living cells, our protocol would be widely used for diverse areas of life sciences.
Sterol Analysis in Kluyveromyces lactis
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.
Molecular Biology
Using CRISPR-ERA Webserver for sgRNA Design
The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/) is one of the state-of-the-art designer webserver tools, which has been developed both for gene editing and gene regulation sgRNA design. This protocol discusses how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.
Neuroscience
Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).
Isolation of Rodent Brain Vessels
The prevalence of neurodegenerative diseases is increasing worldwide. Cerebrovascular disorders and/or conditions known to affect brain vasculature, such as diabetes, are well-known risk factors for neurodegenerative diseases. Thus, the evaluation of the brain vasculature is of great importance to better understand the mechanisms underlying brain damage. We established a protocol for the isolation of brain vessels from rodents. This is a simple, non-enzymatic isolation protocol that allows us to perform comparative studies in different animal models of disease, helping understand the impact of several pathological conditions on brain vasculature and how those alterations predispose to neurodegenerative conditions.
FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction
We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs.
Predator Odor-induced Freezing Test for Mice
The innate fear response is an emotional response that does not require any previously acquired conditioning. One of the standard methods to analyze the innate fear response is a 2,4,5-trimethylthiazoline (TMT)-induced freezing test. TMT is an odor originally isolated from anal secretion of the red fox. Acute TMT exposure has been shown to induce robust freezing behavior in rats and mice (Wallace and Rosen, 2000; Galliot et al., 2012). Here, I show how to expose mice to TMT and how to analyze their freezing behavior.
Plant Science
Isolation and Quantification of Plant Extracellular Vesicles
Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves. The isolated EVs can be quantified using a fluorometric dye to assess total membrane content.
Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants
The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing (Gheysen et al., 1990). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.
Extraction and Molybdenum Blue-based Quantification of Total Phosphate and Polyphosphate in Parachlorella
Inorganic phosphorus is a non-renewable resource and an essential element for life on Earth. Organisms such as algae, protists, and animals can store phosphate (Pi) through uptake of Pi as polyphosphate (poly-P), which is a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. Here, we describe procedures for extraction of total phosphate and poly-P from Parachlorella cells and quantification of orthophosphate based on molybdenum blue assay. The present method may be applicable for other microalgae.
Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to meet the current demands of protein degradation via the UPS within the cell. We describe the adaptation of an intramolecular quenched fluorescence assay utilizing substrate-mimic peptides for the measurement of proteasome activity in total plant extracts. The peptide substrates contain donor-quencher pairs that flank the scissile bond. Following cleavage, the increase in dequenched donor emission of the product is subsequently measured over time and used to calculate the relative proteasome activity.