Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes.
Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes. Methods Mol Biol. 2019;1948:123-143 Authors: Rovere M Abstract α-Synuclein's physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimet...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Rovere M Tags: Methods Mol Biol Source Type: research

In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.
Abstract In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Sanz R, Ovando-Roche P, Udeshi ND, Carr SA, Chung CY, Khurana V Tags: Methods Mol Biol Source Type: research

Expression and Purification of Untagged α-Synuclein.
Expression and Purification of Untagged α-Synuclein. Methods Mol Biol. 2019;1948:261-269 Authors: Powers AE, Patel DS Abstract α-Synuclein (αS) is an abundant neuronal protein which has been implicated, among others, in the pathogenesis of neurodegenerative diseases like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In fact, αS is the major constituent of Lewy bodies, the primarily proteinaceous inclusions found in the nervous tissue of PD and DLB patients. While its physiological role is unclear, it is believed to be involved in the regulation of synaptic vesic...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Powers AE, Patel DS Tags: Methods Mol Biol Source Type: research

Generation and Analysis of Viral Vector-Mediated Rodent Models for Parkinson's Disease.
Abstract The use of viral vector-mediated α-synuclein-overexpressing rodent models for Parkinson's disease has become increasingly accepted to study associated pathology and to use as a platform for therapeutic efficacy testing. We here describe methods to generate such models and how to analyze them by behavioral assessments, histological investigations, and examination of nigrostriatal dopaminergic function. PMID: 30771185 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Karikari AA, Koprich JB, Ip CW Tags: Methods Mol Biol Source Type: research

Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells.
Abstract Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to mon...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Chang CL, Liou J Tags: Methods Mol Biol Source Type: research

Monitoring Non-vesicular Transport of Phosphatidylserine and Phosphatidylinositol 4-Phosphate in Intact Cells by BRET Analysis.
Abstract Non-vesicular lipid transport via lipid transfer proteins (LTPs) at membrane contact sites (MCSs) is critical for the maintenance of the lipid composition of biological membranes. The ability to measure lipid transfer activity of diverse LTPs in live cells without interrupting the fine structural organization is essential to better understand the contribution of non-vesicular lipid transport to membrane organization. Here, we describe a semiquantitative method to analyze phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine (PS) changes at the plasma membrane (PM) as they relate to LTP functions....
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Sohn M, Toth DJ, Balla T Tags: Methods Mol Biol Source Type: research

Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells.
, Várnai P Abstract There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that refle...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Tóth JT, Gulyás G, Hunyady L, Várnai P Tags: Methods Mol Biol Source Type: research

Imaging Lipid Metabolism at the Golgi Complex.
Abstract The development of fluorescence-based molecular imaging has revolutionized cell biology allowing the visualization of specific biomolecules at the microscopic and, more recently, at the nanoscopic scale while in their relevant biological contexts. Nonetheless, despite the imaging toolkit for biologists interested in exploring the subcellular localization and dynamics of proteins and nucleic acids has expanded exponentially in the last decades, the means to visualize and track lipids in cells did not develop to the same extent until recently. Here we described some basic fluorescence-based techniques that ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Capasso S, D'Angelo G Tags: Methods Mol Biol Source Type: research

Advanced In Vitro Assay System to Measure Phosphatidylserine and Phosphatidylethanolamine Transport at ER/Mitochondria Interface.
Abstract A number of previous studies have shown that phospholipid molecules come and go between the endoplasmic reticulum (ER) and mitochondrial membranes while the molecular basis of non-vesicular phospholipid transport is still not understood well. In this chapter, we describe an optimized method that uses membrane fractions isolated from yeast cells to directly analyze phospholipid transport between the ER and mitochondria. With this assay, we are able to assess not only the ER-to-mitochondria but also mitochondria-to-ER transports at the same time. We believe that this assay system can accelerate the research...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Tamura Y, Kojima R, Endo T Tags: Methods Mol Biol Source Type: research

Measurement of Lipid Transport in Mitochondria by the MTL Complex.
Abstract Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This li...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Jouhet J, Gros V, Michaud M Tags: Methods Mol Biol Source Type: research

Bi- and Trifunctional Lipids for Visualization of Sphingolipid Dynamics within the Cell.
r D Abstract Investigations into lipid localization and transport are often hampered by a lack of methods and tools to faithfully visualize lipids in the context of living cells, since fluorescent modifications drastically change lipid properties. Here, we describe the use of bifunctional as well as trifunctional sphingosine to reveal its subcellular localization via crosslinking, fixation, and specific staining by click reaction with a fluorophore. Additionally, these probes allow investigations into lipid metabolism as revealed by thin-layer chromatography. PMID: 30790251 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Höglinger D Tags: Methods Mol Biol Source Type: research

Measurement of Intracellular Sterol Transport in Yeast.
Abstract Intracellular sterol transport occurs largely by non-vesicular mechanisms in which sterol transport proteins extract sterol from one membrane and transfer it to another across the cytoplasm. Here we describe a suite of complementary assays to measure intracellular sterol transport in the model eukaryote Saccharomyces cerevisiae, as well as to quantify protein-mediated sterol transport between populations of vesicles in vitro. The in vivo assays can be adapted to study sterol transport in other cell types. PMID: 30790253 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Chauhan N, Jentsch JA, Menon AK Tags: Methods Mol Biol Source Type: research

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.
Abstract Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at th...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Wilhelm LP, Voilquin L, Kobayashi T, Tomasetto C, Alpy F Tags: Methods Mol Biol Source Type: research

Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein.
Abstract Mammalian cells carefully control their cholesterol levels by employing multiple feedback mechanisms to regulate synthesis of cholesterol and uptake of cholesterol from circulating lipoproteins. Most of a cell's cholesterol (~80% of total) is in the plasma membrane (PM), but the protein machinery that regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell's cholesterol. How does the ER communicate with PM to monitor cholesterol levels in that membrane? Here, we describe a tool, ALOD4, that helps us answer this questi...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Endapally S, Infante RE, Radhakrishnan A Tags: Methods Mol Biol Source Type: research

Measurement of Lysophospholipid Transport Across the Membrane Using Escherichia coli Spheroplasts.
Abstract In the inner membrane of Gram-negative bacteria lysophospholipid transporter (LplT) and the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase (Aas) form a glycerophospholipid remodeling system, which is capable of facilitating rapid retrograde translocation of lyso forms of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin across the cytoplasmic membrane. This coupled remodeling enzyme tandem provides an effective method for the measurement of substrate specificity of the lipid regeneration and lysophospholipid transport per se across t...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Lin Y, Zheng L, Bogdanov M Tags: Methods Mol Biol Source Type: research

In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.
Abstract The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial lipid-binding Protein (SMP) domain, a lipid-harboring module through which they mediate lipid transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and lipid transport functions of E-Syt1. Its membrane tethering activity is monitored through ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Bian X, De Camilli P Tags: Methods Mol Biol Source Type: research

In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.
Abstract Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and accept...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Kenoth R, Brown RE, Kamlekar RK Tags: Methods Mol Biol Source Type: research

In Vitro Strategy to Measure Sterol/Phosphatidylinositol-4-Phosphate Exchange Between Membranes.
Abstract Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for phosphatidylinositol-4-phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificia...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Lipp NF, Drin G Tags: Methods Mol Biol Source Type: research

Synthesis of Fluorescent Membrane-Spanning Lipids for Studies of Lipid Transfer and Membrane Fusion.
Abstract For uncompromised in vitro assays for intermembrane lipid transfer and membrane fusion fluorescent membrane-spanning lipids have proved to be invaluable tools. These lipids in contrast to phosphoglycerolipids and sphingolipids are resistant to spontaneous as well as protein-mediated intermembrane transfer. Here I describe the synthesis of some homo-substituted fluorescent bipolar membrane-spanning lipids that bear a fluorescent tag either directly or via a phosphoethanolamine spacer to the lipid core. For the synthesis the lipid core of the bipolar membrane-spanning lipids, i.e., the tetraether lipid cald...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Schwarzmann G Tags: Methods Mol Biol Source Type: research

A Stable Luciferase Reporter System to Characterize LXR Regulation by Oxysterols and Novel Ligands.
Abstract Nuclear receptors (NRs) are ligand-activated transcription factors. Class 2 NRs, such as the liver X receptor (LXR)α and (LXR)β, are typically retained in the nucleus bound to the DNA in both the presence and absence of ligand. Upon binding ligands including hydroxylated cholesterol, LXR releases corepressor proteins in exchange for coactivators resulting in target gene transcription. Activity of the LXRs therefore depends on a combination of the local ligand concentration(s) and cofactor expression, which itself is a function of cell and tissue type, mutation load, and epigenetic regulation. C...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Hutchinson SA, Thorne JL Tags: Methods Mol Biol Source Type: research

Isolation and Analysis of Human Monocytes and Adipose Tissue Macrophages.
Abstract Monocytes and macrophages are cells of the innate immune system, existing in circulation and permeating every tissue of the human body. These cells represent the first responders to stress, and their inflammatory activation forms part of virtually every human disease. It is for these reasons that several approaches have been developed in order to phenotypically and functionally analyze these cells and their subpopulations. These inflammatory cells have been consistently demonstrated to undergo inflammatory polarization through the engagement of lipid-activated nuclear receptors, namely, through PPARs and ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Julla JB, Ballaire R, Ejlalmanesh T, Gautier JF, Venteclef N, Alzaid F Tags: Methods Mol Biol Source Type: research

Isolation and Purification of Tissue Resident Macrophages for the Analysis of Nuclear Receptor Activity.
Abstract Tissue resident macrophages (TRMs) are multifunctional immune cells present in all tissues, contributing to the correct development, homeostasis, and protection against pathogens and injury. TRMs are morphologically and functionally heterogeneous, as a result of both the diversity of tissue environments in which they reside and their complex origin. Furthermore, some specific TRM populations are controlled by nuclear receptors. A widely used method for studying the role of nuclear receptors in immune cells is flow cytometry. Although flow cytometry is extensively used in tissues such as the periphera...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Alonso-Herranz L, Porcuna J, Ricote M Tags: Methods Mol Biol Source Type: research

Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells.
Castrillo A Abstract Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRβ). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differenti...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Ramón-Vázquez A, de la Rosa JV, Tabraue C, Castrillo A Tags: Methods Mol Biol Source Type: research

Dual Cross-Linking Chromatin Immunoprecipitation Protocol for Next-Generation Sequencing (ChIPseq) in Macrophages.
Abstract Macrophages arise from distinct progenitor cell populations throughout development and are one of the most diverse cell types, capable of performing discrete functions, undergoing distinct modes of activation, and infiltrating or residing in numerous niches in the body. In adapting to their environments, macrophages display high levels of plasticity which is associated with profound epigenomic and transcriptional changes. Understanding these changes has been greatly facilitated by the next-generation sequencing (NGS)-based approaches such as RNAseq and chromatin immunoprecipitation (ChIP)seq. Despite the ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Rollins DA, Rogatsky I Tags: Methods Mol Biol Source Type: research

Quantifying Cellular Cholesterol Efflux.
Abstract Measuring cholesterol efflux involves the tracking of cholesterol movement out of cells. Cholesterol efflux is an essential mechanism to maintain cellular cholesterol homeostasis, and this process is largely regulated via the LXR transcription factors and their regulated genes, the ATP-binding cassette (ABC) cholesterol transporters ABCA1 and ABCG1. Typically, efflux assays are performed utilizing radiolabeled cholesterol tracers to label intracellular cholesterol pools, and these assays may be tailored to quantify the efflux of exogenously delivered cholesterol or alternatively the efflux of newly synthe...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Robichaud S, Ouimet M Tags: Methods Mol Biol Source Type: research

Methods for Assessing the Effects of LXR Agonists on Macrophage Bacterial Infection.
or AF Abstract Macrophages are phagocytic cells that actively engulf and kill microorganisms within a specialized phagolysosomal system. Several pathogenic bacteria, however, actively co-opt host mechanisms and escape from microbial digestion to establish intracellular replication within macrophages. This chapter highlights detailed protocols to measure the effects of the LXR pathway on bacterial infection of murine bone marrow-derived macrophages. PMID: 30825149 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Glaría E, Matalonga J, Valledor AF Tags: Methods Mol Biol Source Type: research

Measuring Apoptotic Cell Engulfment (Efferocytosis) Efficiency.
Abstract Efferocytosis is the process of recognizing and removing dead and dying cells, performed by a variety of phagocytic cells including macrophages. It has recently been shown that liver X receptor (LXR) signaling in macrophages regulates the expression of important efferocytosis receptors, bridging and signaling molecules. Here we describe a sensitive yet robust efferocytosis assay, optimized to measure bone marrow-derived macrophage (BMDM) apoptotic cell engulfment capability. This assay can be applied to genetically or pharmacologically altered BMDMs. PMID: 30825150 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Gage MC Tags: Methods Mol Biol Source Type: research

Methods to Study Monocyte and Macrophage Trafficking in Atherosclerosis Progression and Resolution.
Abstract Monocytes are circulating cells imperative to the response against pathogens. Upon infection, they are quickly recruited to the affected tissue where they can differentiate into specialized phagocytes and antigen-presenting cells. Additionally, monocytes play a vital role in chronic inflammation, where they can promote and enhance inflammation or induce its resolution. There are two major subsets of monocytes, "inflammatory" and "nonclassical," which are believed to have distinct functions. In atherosclerosis, both types of monocytes are constantly recruited to lesions, where they cont...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Weinstock A, Fisher EA Tags: Methods Mol Biol Source Type: research

Analyzing T-Cell Plasma Membrane Lipids by Flow Cytometry.
We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane "lipid order" using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types. PMID: 30825155 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Waddington KE, Pineda-Torra I, Jury EC Tags: Methods Mol Biol Source Type: research

The structural and gene expression hypotheses in laminopathic diseases-not so different after all.
Abstract Laminopathies are a diverse group of rare diseases with various pathologies in different tissues, which are linked to mutations in the LMNA gene. Historically, the structural disease model proposed mechanical defects of the lamina and nuclear fragility, the gene expression model impairment of spatial chromatin organization and signaling pathways as underlying mechanisms leading to the pathologies. Exciting findings in the past few years showing that mechanical forces are directly transmitted into the nucleus, where they affect chromatin organization and mechanoresponsive signaling molecules, have led to a...
Source: Mol Biol Cell - July 15, 2019 Category: Molecular Biology Authors: Osmanagic-Myers S, Foisner R Tags: Mol Biol Cell Source Type: research

Cytoskeletal proteins in the cell nucleus: a special nuclear actin perspective.
Abstract The emerging role of cytoskeletal proteins in the cell nucleus has become a new frontier in cell biology. Actin and actin-binding proteins regulate chromatin and gene expression, but importantly they are beginning to be essential players in genome organization. These actin-based functions contribute to genome stability and integrity while affecting DNA replication and global transcription patterns. This is likely to occur through interactions of actin with nuclear components including nuclear lamina and subnuclear organelles. An exciting future challenge is to understand how these actin-based genome-wide ...
Source: Mol Biol Cell - July 15, 2019 Category: Molecular Biology Authors: Percipalle P, Vartiainen M Tags: Mol Biol Cell Source Type: research

Optimizing the Cell Culture Microenvironment.
Abstract The survival, proliferation, and differentiation of cells in culture are determined not only by their intrinsic potential but also by cues provided by the permissive or restrictive microenvironment in which they reside. The robustness and reproducibility of cell culture assays and endpoints relies on the stability of that microenvironment and vigilant attention to the control of variables that affect cell behavior during culture. These often underappreciated variables include, but are not limited to, medium pH and buffering, osmolarity, composition of the gas phase, the timing and periodicity of refeeding...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Bertoncello I Tags: Methods Mol Biol Source Type: research

Propagation and Maintenance of Mouse Embryonic Stem Cells.
Abstract Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irr...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Paynter JM, Chen J, Liu X, Nefzger CM Tags: Methods Mol Biol Source Type: research

Production of High-Titer Lentiviral Particles for Stable Genetic Modification of Mammalian Cells.
Abstract Lentiviral gene transfer technologies exploit the natural efficiency of viral transduction to integrate exogenous genes into mammalian cells. This provides a simple research tool for inducing transgene expression or endogenous gene knockdown in both dividing and nondividing cells. This chapter describes an improved protocol for polyethylenimine (PEI)-mediated multi-plasmid transfection and polyethylene glycol (PEG) precipitation to generate and concentrate lentiviral vectors. PMID: 30788817 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Larcombe MR, Manent J, Chen J, Mishra K, Liu X, Nefzger CM Tags: Methods Mol Biol Source Type: research

Generation of Mouse-Induced Pluripotent Stem Cells by Lentiviral Transduction.
Abstract Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of four transcription factors: Oct4, Klf4, Sox2, and c-Myc (OKSM). These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) t...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Liu X, Chen J, Firas J, Paynter JM, Nefzger CM, Polo JM Tags: Methods Mol Biol Source Type: research

Identification of Circulating Endothelial Colony-Forming Cells from Murine Embryonic Peripheral Blood.
Abstract Human umbilical cord blood contains highly proliferative circulating endothelial colony-forming cells (ECFC). These cells have promising therapeutic potential for various cardiovascular diseases by possessing robust in vitro clonal expansion potential and the ability to form functional blood vessels in vivo upon transplantation into recipient immunodeficient mice. However whether similar cells also exist in murine blood remains unresolved, which impedes the study of circulating ECFC biology using murine models. Here we describe a method to identify and culture murine embryonic peripheral blood-derived cir...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Lin Y, Gil CH, Yoder MC Tags: Methods Mol Biol Source Type: research

Imaging and Analysis of Mouse Embryonic Whole Lung, Isolated Tissue, and Lineage-Labelled Cell Culture.
Abstract Research on lung development and disease frequently utilizes mouse models to conduct in vitro experiments. Such experiments involve multiple methodologically distinct stages, from careful consideration of mouse models used to obtain biological samples, to the culturing and imaging of those samples, and finally, to post-imaging analysis. Here, we detail basic protocols to assist with each of these stages. First, we discuss harvesting and preparing biological samples; second, we focus on culturing embryonic whole lung explants and isolated mesenchyme and epithelium; third, we specify the basics of obtaining...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Jones M, Bellusci S Tags: Methods Mol Biol Source Type: research

Mouse Hematopoietic Stem Cell Modification and Labelling by Transduction and Tracking Posttransplantation.
Abstract The tracking of the hematopoietic potential of genetically manipulated fluorescent hematopoietic stem cells (HSC) in the bone marrow (BM) allows the assessment of regulatory processes involved in the re-establishment of hematopoiesis posttransplant. Herein, we describe the means to assess the consequence of expressing specific genes in HSC on their engraftment potential posttransplant. PMID: 30788822 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Cao B, Li S, Pritchard C, Williams B, Nilsson SK Tags: Methods Mol Biol Source Type: research

Genetic Manipulation and Selection of Mouse Mesenchymal Stem Cells for Delivery of Therapeutic Factors In Vivo.
Abstract Bone marrow-derived mesenchymal stem cells (MSCs) hold great potential as an ex vivo cellular system for delivery of therapeutic proteins to the diseased or damaged central nervous system (CNS). This adult stem cell population has considerable translational potential for autologous transplantation due to lack of ethical concerns, accessibility, multipotent nature, and plasticity. Here we describe a methodology and outline a strategy using lentiviral vectors for producing lines of MSCs hypersecreting neurotrophic growth factors (e.g., brain-derived neurotrophic factor (BDNF) and/or glial cell line-derived ...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Sakaguchi DS Tags: Methods Mol Biol Source Type: research

Isolation and Culture of Primary Mouse Middle Ear Epithelial Cells.
Abstract Epithelial abnormalities underpin the development of the middle ear disease, otitis media (OM). Until now, a well-characterized in vitro model of the middle ear (ME) epithelium that replicates the complex cellular composition of the middle ear has not been available. This chapter describes the development of a novel in vitro model of mouse middle ear epithelial cells (mMECs), cultured at the air-liquid interface (ALI). This system enables recapitulation of the characteristics of the native murine ME epithelium. We demonstrate that mMECs undergo differentiation into the varied cell populations seen within ...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Mulay A, Akram K, Bingle L, Bingle CD Tags: Methods Mol Biol Source Type: research

Isolation and Propagation of Lacrimal Gland Putative Epithelial Progenitor Cells.
We present a protocol for isolation of putative epithelial progenitor cells from mouse lacrimal gland (LG) by fluorescence-activated cell sorting (FACS). Isolated LG epithelial progenitor cells can be cultured as 3D reaggregates within extracellular matrix gel or plated as a monolayer. 3D cultures could be maintained for several days and then dissociated with trypsin and plated as monolayer cultures, processed for analysis (e.g., mRNA/protein expression) and/or used for transplantations. Our goal is to provide researchers with a method that can be used as is or modified if isolation of other LG epithelial cell types is req...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Makarenkova HP, Meech R Tags: Methods Mol Biol Source Type: research

Organotypic Culture of Adult Mouse Retina.
r B Abstract Retinal explant culture systems have the potential to mimic the functional dynamics of the organ beyond those of the dissociated cells, thus making this technique a very powerful intermediate model system between in vitro cell cultures and in vivo animal models. The different retinal layers made of highly specialized cell types remain intact, while glia cell reactions and/or intercellular interactions can be evaluated under well-defined conditions in the lab.In retinal disorders neurodegeneration of mature retinal cells takes place. Therefore, we investigated the adult murine neuroretina in organ cult...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Müller B Tags: Methods Mol Biol Source Type: research

Langendorff-Free Isolation and Propagation of Adult Mouse Cardiomyocytes.
Abstract Isolation of healthy, intact cardiomyocytes from the adult mouse heart for cardiac research is challenging. Traditional protocols depend upon specialized Langendorff apparatus, which requires extensive training and presents significant technical and logistical barriers. Described here is a much simplified technique, introducing optimized dissociation buffers to the heart by direct needle injection into the left ventricle, allowing deep myocardial perfusion and the isolation of high yields of viable, rod-shaped cardiomyocytes, using only standard surgical and laboratory equipment. This method also permits ...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Ackers-Johnson M, Foo RS Tags: Methods Mol Biol Source Type: research

Isolation, Culture, and Characterization of Primary Mouse Epidermal Keratinocytes.
Abstract Epidermis, the outermost layer of the skin, plays a critical role as both a physical and immunological barrier protecting the internal tissues from external environmental insults, such as pathogenic bacteria, fungi, viruses, UV irradiation, and water loss. Epidermal keratinocytes (KC), the predominant cell type in the skin epidermis, are in the front line of skin defense. Here we describe methods to isolate and culture primary epidermal KC from neonatal and adult mouse skin and describe in vitro assays to study and characterize KC proliferation and differentiation and pro-inflammatory responses to viral p...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Zhang LJ Tags: Methods Mol Biol Source Type: research

Isolation and Propagation of Mammary Epithelial Stem and Progenitor Cells.
Abstract Several methods of mammary gland dissociation have been described that utilize a combined strategy of mechanical and enzymatic dissociation to isolate mammary epithelial cells (MECs) from intact tissue (Smalley et al., J Mammary Gland Biol Neoplasia 17:91-97, 2012). Here we detail a robust method that enables the isolation of all major stem and progenitor MEC populations, which has been successfully used to study stem cell behavior when coupled with transplantation and in vitro assays (Shackleton et al., Nature 439:84-88, 2006; Bouras et al., Cell Stem Cell 3:429-441, 2008; Sheridan et al., BMC Cancer 15:...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Sheridan JM, Visvader JE Tags: Methods Mol Biol Source Type: research

An Organoid Assay for Long-Term Maintenance and Propagation of Mouse Prostate Luminal Epithelial Progenitors and Cancer Cells.
Abstract Historically, prostate luminal epithelial progenitors and cancer cells have been difficult to culture, thus hampering the generation of representative models for the study of prostate homeostasis, epithelial lineage hierarchy relationship and cancer drug efficacy assessment. Here, we describe a newly developed culture methodology that can efficiently grow prostate luminal epithelial progenitors and cancer cells as organoids. Notably, the organoid assay favors prostate luminal cell growth, thus minimizing basal cell dominance upon the establishment and continuous propagation of prostate epithelial cells. I...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Shu Y, Chua CW Tags: Methods Mol Biol Source Type: research

Isolation, Purification, and Culture of Mouse Pancreatic Islets of Langerhans.
e;s N Abstract Pancreatic islets constitute an important tool for research and clinical applications in the field of diabetes. They are used for transplantation, unraveling new mechanisms in insulin secretion, studying pathophysiological pathways in diseased cells, and pharmacological research aimed at developing improved therapeutic strategies. Therefore, fine-tuning islet isolation protocols remains an important objective for reliable investigations. Here we describe a relatively simple mouse islet isolation protocol that relies on enzymatic digestion using low-activity collagenase and several sedimentation and ...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Saliba Y, Farès N Tags: Methods Mol Biol Source Type: research

Identification and In Vitro Expansion of Adult Hepatocyte Progenitors from Chronically Injured Livers.
Abstract The liver performs a number of physiologically important functions. Hepatocytes are the liver parenchymal cells performing most of those functions. Therefore, it is important to recover functional hepatocytes after hepatic injury and prepare a mass of hepatocytes for regenerative medicine. We have found that mature hepatocytes dedifferentiate to hepatocyte progenitors in chronically injured mouse liver. Those hepatocyte progenitors can be isolated as CD24+EpCAM- cells from the CD31-CD45- fraction, which clonally proliferate and efficiently re-differentiate to functional hepatocytes both in vitro and in vi...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Tanimizu N Tags: Methods Mol Biol Source Type: research

The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential.
gner DE Abstract Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unp...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Bölükbas DA, De Santis MM, Alsafadi HN, Doryab A, Wagner DE Tags: Methods Mol Biol Source Type: research

Mouse Lung Tissue Slice Culture.
Abstract Precision-cut lung slices (PCLS) represent an ex vivo model widely used in visualizing interactions between lung structure and function. The major advantage of this technique is that the presence, differentiation state, and localization of the more than 40 cell types that make up the lung are in accordance with the physiological situation found in lung tissue, including the right localization and patterning of extracellular matrix elements. Here we describe the methodology involved in preparing and culturing PCLS followed by detailed practical information about their possible applications. PMID: 3078...
Source: Mol Biol Cell - July 13, 2019 Category: Molecular Biology Authors: Wu X, van Dijk EM, Bos IST, Kistemaker LEM, Gosens R Tags: Methods Mol Biol Source Type: research