Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.
Abstract Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src sy...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Sato KI, Tokmakov AA Tags: Methods Mol Biol Source Type: research

Embryological and Genetic Manipulation of Chick Development.
Abstract The ability to combine embryological manipulations with gene function analysis in an amniote embryo makes the chick a valuable system for the vertebrate developmental biologist. This chapter describes methods for those unfamiliar with the chick system wishing to initiate experiments in their lab. After outlining methods to prepare chick embryos, protocols are provided for introducing beads or cells expressing secreted factors, and for culturing tissue explants as a means of assessing development in vitro. Approaches to achieve gain of function and loss of function (morpholino oligonucleotides) in chick ar...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Gammill LS, Jacques-Fricke B, Roffers-Agarwal J Tags: Methods Mol Biol Source Type: research

RNA Tomography for Spatially Resolved Transcriptomics (Tomo-Seq).
Abstract Embryonic development is heavily dependent on temporally and spatially restricted gene expression. Spatially resolved measurements of gene expression are therefore crucial for identifying novel regulators and the understanding of their function. However, in situ methods do not resolve global gene expression, and sequencing-based methods usually do not provide spatial information. Here, we describe tomo-seq, a method that combines classical histological sectioning of embryos or tissues with a highly sensitive RNA-sequencing technique. Application of tomo-seq to zebrafish embryos allows reconstructing ...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Holler K, Junker JP Tags: Methods Mol Biol Source Type: research

Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos.
Abstract This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an "anchor" protein and a "bait" protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117-126, 2016) and describe the imaging parameters ...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Buckley CE Tags: Methods Mol Biol Source Type: research

Reptile Embryology and Regeneration.
Abstract Reptiles (lizards, snakes, turtles, and crocodilians) are becoming increasingly popular as models for developmental investigations. In this review the leopard gecko, Eublepharis macularius, is presented as a reptilian model for embryonic and tissue regeneration studies. We provide details of husbandry and breeding and discuss aspects of embryonic nutrition, egg anatomy, and sex determination. We provide comprehensive protocols for transcardial perfusion, short-term anesthesia using the injectable anesthetic Alfaxan, and full-thickness cutaneous biopsy punches, used in geckos for the study of scar-free wou...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Vickaryous MK, Gilbert EAB Tags: Methods Mol Biol Source Type: research

Manipulation of Developmental Function in Turtles with Notes on Alligators.
We describe protocols for the preparation of turtle embryos for ex ovo culture, electroporation, in situ hybridization, and microcomputed tomography. Because these protocols have been adapted and optimized from methods used for frog, chick, and mouse embryos, it is likely that they could be used for other reptilian species. Notes are included for alligator embryos where appropriate. PMID: 30737695 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Moustakas-Verho JE, McLennan R, Spengler J, Kulesa PM, Cebra-Thomas JA Tags: Methods Mol Biol Source Type: research

Methods for Isolating the Balbiani Body/Germplasm from Xenopus laevis Oocytes.
We describe here two techniques to isolate the Bb/germplasm from Xenopus laevis primary oocytes. PMID: 30737696 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Butler A, Owens D, King ML, Aguero T Tags: Methods Mol Biol Source Type: research

Visualizing the Balbiani Body in Zebrafish Oocytes.
Abstract Approaches to visualize the Balbiani body of zebrafish primary oocytes using protein, RNA, and mitochondrial markers are described. The method involves isolation, histology, staining, and microscopic examination of early zebrafish oocytes. These techniques can be applied to visualize gene products that are localized to the Balbiani body, and when applied to mutants can be used to decipher molecular and genetic pathways acting in Balbiani body development in early oocytes. PMID: 30737697 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Lee KL, Marlow FL Tags: Methods Mol Biol Source Type: research

Isolation of Zebrafish Balbiani Bodies for Proteomic Analysis.
Abstract Proteomic characterization of isolated organelles can provide insight into the functional components of the structure and novel targets for further testing. Germplasm in developing oocytes is difficult to isolate for protein identification because not all types of germplasm are stable outside of the cytoplasm. In zebrafish, the Balbiani body forms a proteinaceous aggregate that contains the germplasm and we found is stable outside of the oocyte. Here we present a manual isolation protocol that collects intact Balbiani bodies from stage I zebrafish oocytes. We lysed oocytes by passing them through a syring...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Jamieson-Lucy A, Mullins MC Tags: Methods Mol Biol Source Type: research

Generation of Xenopus Haploid, Triploid, and Hybrid Embryos.
Abstract Frog species of the genus Xenopus are widely used for studies of cell and developmental biology, and recent genome sequencing has revealed interesting phylogenetic relationships. Here we describe methods to generate haploid, triploid, and hybrid species starting from eggs and sperm of Xenopus laevis and Xenopus tropicalis that enable investigation of how genome size and content affect physiology at the organismal, cellular, and subcellular levels. PMID: 30737699 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Gibeaux R, Heald R Tags: Methods Mol Biol Source Type: research

Germ Cell Transplantation in Avian Species.
Abstract Germ cell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germ cell transplantation systems have been developed, including sperm, oocyte, or germline stem cell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germ cell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos. PMID: 30737700 [PubM...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Park YH, Kim YM, Han JY Tags: Methods Mol Biol Source Type: research

Production of Germ-Line Chimeras in Zebrafish.
Abstract The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct i...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Saito T, Goto R, Rivers N, Yamaha E Tags: Methods Mol Biol Source Type: research

Optimized Protocol of Zebrafish Somatic Cell Nuclear Transfer (SCNT).
Abstract Zebrafish (Danio rerio) is an established animal model to study developmental biology as well as a wide array of human diseases. Here we describe a protocol for somatic cell nuclear transfer (SCNT). This protocol can be used to introduce genetic modifications in zebrafish and for the study of cell plasticity. PMID: 30737703 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Prukudom S, Siripattarapravat K, Poulos W, Cibelli JB Tags: Methods Mol Biol Source Type: research

Modeling Embryonic Cleavage Patterns.
Abstract The division patterns of early invertebrate and vertebrate embryos are key to the specification of cell fates and embryo body axes. We here describe a generic computational modeling method to quantitatively test mechanisms which specify successive division position and orientation of eggs and early blastomeres in 3D. This approach should serve to motivate and guide future experimental work on the mechanisms controlling early embryo morphogenesis. PMID: 30737705 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Ershov D, Minc N Tags: Methods Mol Biol Source Type: research

Tailoring N-Glycan Biosynthesis for Production of Therapeutic Proteins in Saccharomyces cerevisiae.
Abstract The ability to control and adjust the N-glycosylation pathway of Saccharomyces cerevisiae is a key step toward production of therapeutic glycoproteins such as antibodies or erythropoietin. The focus of this chapter is to describe the road from yeast-type N-glycosylation to human-type complex N-glycosylation. The chapter describes the cell engineering and provides the detailed analytical procedures required to perform glycan analysis using MALDI-TOF mass spectrometry. PMID: 30737743 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Piirainen MA, Salminen H, Frey AD Tags: Methods Mol Biol Source Type: research

Improving the Production of Cofactor-Containing Proteins: Production of Human Hemoglobin in Yeast.
vic D Abstract Human hemoglobin is an essential protein, whose main function as an oxygen carrier is indispensable for life. Hemoglobin is a cofactor-containing protein with heme as prosthetic group. Same as in humans, heme is synthesized in many organisms in a complex pathway involving two cellular compartments (mitochondria and cytosol), which is tightly regulated. Red blood cells (erythrocytes) are specialized and adapted for production and transport of the hemoglobin molecules. In addition to oxygen binding, hemoglobin can participate in a variety of chemical reactions by its iron and heme and may become ...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Ishchuk OP, Martínez JL, Petranovic D Tags: Methods Mol Biol Source Type: research

Membrane Protein Production in Yeast: Modification of Yeast Membranes for Human Membrane Protein Production.
Abstract Approximately 30% of the genes in the human genome code for membrane proteins, and yet we know relatively little about these complex molecules. Therefore, the biochemical and structural characterization of this challenging class of proteins represents an important frontier in both fundamental research and advances in drug discovery. However, due to their unique physical properties and requirement for association with cellular membranes, expression in heterologous systems is often daunting. In this chapter we describe how to engineer the yeast Pichia pastoris to obtain humanized sterol compositions. By imp...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Emmerstorfer-Augustin A, Wriessnegger T, Hirz M, Zellnig G, Pichler H Tags: Methods Mol Biol Source Type: research

Yeast Surface Display and Cell Sorting of Antigen-Binding Fc Fragments.
k-Knopp G Abstract Since the introduction of the yeast display platform, this method has increasingly gained popularity for the discovery and affinity maturation of antibodies and other protein scaffolds intended for antigen recognition. Yeast display is particularly well suited for the selection of antigen-binding Fc fragments (Fcabs) as it allows rapid combinatorial library construction via gap repair-driven homologous recombination and an efficient display of a glycosylated Fc able to interact with Fcγ receptors. Apart from expression-related normalization, isolation of properly folded Fcabs can be guided...
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Sádio F, Stadlmayr G, Stadlbauer K, Rüker F, Wozniak-Knopp G Tags: Methods Mol Biol Source Type: research

Primary Recovery of Yeast Culture Supernatant for Recombinant Protein Purification.
Abstract The harvest of a bench scale high cell density fermentation of Pichia pastoris, using standard laboratory equipment, to process products from supernatant is described. The process consists of a centrifugation step and a depth filtration, followed by a (sterile) membrane filtration. The procedure yields supernatant ready for the next purification step such as chromatography. PMID: 30737749 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 20, 2019 Category: Molecular Biology Authors: Maurer MM, Schillinger H Tags: Methods Mol Biol Source Type: research

Mol Biol Cell; +111 new citations
111 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: Mol Biol Cell These pubmed results were generated on 2019/06/19PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites. (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 19, 2019 Category: Molecular Biology Tags: Report Source Type: research

Generation of Neural Stem Cells from Induced Pluripotent Stem Cells.
Abstract Neural stem cells (NSCs) are defined by three necessary but not sufficient criteria: (1) self-renewable, (2) ability to generate a large number of progeny, and (3) ability to differentiate into the principal central nervous system (CNS) cell types, neurons, astrocytes, and oligodendrocytes. There are various approaches to derive neural lineages from pluripotent stem cells. It is well recognized that the chosen method of NSC derivation is critical to answering the basic biology question under investigation, to the success rate in drug discovery and to the efficacy of the therapeutic cells intended for repa...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Daadi MM Tags: Methods Mol Biol Source Type: research

Making NSC and Neurons from Patient-Derived Tissue Samples.
We describe a method of deriving NSCs and differentiated mature neurons highlighting key steps of the core protocol. We also provide detailed information of the transcription factor and morphogen map of the developing brain which can be used as a guide to derive region- and lineage-specific NSCs and differentiated neurons. PMID: 30656618 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Mukherjee O, Acharya S, Rao M Tags: Methods Mol Biol Source Type: research

Generation of Definitive Neural Progenitor Cells from Human Pluripotent Stem Cells for Transplantation into Spinal Cord Injury.
Abstract In this chapter, we first describe two interchangeable protocols optimized in our lab for deriving definitive neuronal progenitor cells from human pluripotent stem cells (hPSCs). The resultant NPCs can then be propagated and differentiated to produce differing proportions of neurons, oligodendrocytes, and astrocytes as required for in vitro cell culture studies or in vivo transplantation. Following these protocols, we explain the method for transplanting these cells into the rat model of spinal cord injury (SCI). PMID: 30656619 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Khazaei M, Ahuja CS, Rodgers CE, Chan P, Fehlings MG Tags: Methods Mol Biol Source Type: research

Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells.
Abstract We have previously shown that human parthenogenetic stem cells (hpSC) can be chemically directed to differentiate into a homogeneous population of multipotent neural stem cells (hpNSC) that are scalable, cryopreservable, express all the appropriate neural markers, and can be further differentiated into functional dopaminergic neurons. Differentiation of hpSC into hpNSC provides a platform to study the molecular basis of human neural differentiation, to develop cell culture models of neural disease, and to provide neural stem cells for the treatment of neurodegenerative diseases. Additionally, the hpNSC th...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Gonzalez R, Garitaonandia I, Semechkin A, Kern R Tags: Methods Mol Biol Source Type: research

Chemically Defined Neural Conversion of Human Pluripotent Stem Cells.
il; I Abstract Human pluripotent stem cells (hPSCs) are characterized by their ability to self-renew and differentiate into any cell type of the human body. To fully utilize the potential of hPSCs for translational research and clinical applications, it is critical to develop rigorous cell differentiation protocols under feeder-free conditions that are efficient, reproducible, and scalable for high-throughput projects. Focusing on neural conversion of hPSCs, here we describe robust small molecule-based procedures that generate neural stem cells (NSCs) in less than a week under chemically defined conditions. These ...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Chen Y, Tristan CA, Mallanna SK, Ormanoglu P, Titus S, Simeonov A, Singeç I Tags: Methods Mol Biol Source Type: research

In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology.
Abstract Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or ...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Hansen MG, Tornero D, Canals I, Ahlenius H, Kokaia Z Tags: Methods Mol Biol Source Type: research

Differentiation of Neural Stem Cells Derived from Induced Pluripotent Stem Cells into Dopaminergic Neurons.
Abstract Dopaminergic (DA) neurons are involved in many critical functions within the central nervous system (CNS), and dopamine neurotransmission impairment underlies a wide range of disorders from motor control deficiencies, such as Parkinson's disease (PD), to psychiatric disorders, such as alcoholism, drug addictions, bipolar disorders, schizophrenia and depression. Neural stem cell-based technology has potential to play an important role in developing efficacious biological and small molecule therapeutic products for disorders with dopamine dysregulation. Various methods of differentiating DA neuron...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Daadi MM Tags: Methods Mol Biol Source Type: research

Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells.
We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening. PMID: 30656624 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Tofoli FA, Semeano ATS, Oliveira-Giacomelli Á, Gonçalves MCB, Ferrari MFR, Veiga Pereira L, Ulrich H Tags: Methods Mol Biol Source Type: research

Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.
Abstract Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique. PMID: 30656625 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Hong H, Daadi MM Tags: Methods Mol Biol Source Type: research

Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells.
Abstract Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP b...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Kim J, Daadi MM Tags: Methods Mol Biol Source Type: research

Bioinformatics Analysis of Single-Cell RNA-Seq Raw Data from iPSC-Derived Neural Stem Cells.
Abstract This chapter describes a pipeline for basic bioinformatics analysis of single-cell sequencing data (see Chap. 10 : Single-Cell Library Preparation). Starting with raw sequencing data, we describe how to quality check samples, to create an index from a reference genome, to align the sequences to an index, and to quantify transcript abundances. The curated data sets will enable differential expression analysis, population analysis, and pathway analysis. PMID: 30656627 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Kim J, Daadi MM Tags: Methods Mol Biol Source Type: research

Assay for Assessing Mitochondrial Function in iPSC-Derived Neural Stem Cells and Dopaminergic Neurons.
Abstract Rapid and reliable assessment of mitochondrial bioenergetics is a vital tool in drug discovery studies aimed at reversing or improving mitochondrial dysfunction. Induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) carry and replicate the donor disease pathology and can be an ideal cellular model for phenotypic screening of compounds. Herein we describe the use of Seahorse XFe96 analyzer to assess mitochondrial functions in iPSC-derived NSCs for drug screening. PMID: 30656628 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Roy-Choudhury G, Daadi MM Tags: Methods Mol Biol Source Type: research

Reference Transcriptome for Deriving Marmoset  Induced Pluripotent Stem Cells.
Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells. Methods Mol Biol. 2019;1919:175-186 Authors: Yang G, Hong H, Torres A, Malloy KE, Roy-Choudhury G, Kim J, Daadi MM Abstract Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for researc...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Yang G, Hong H, Torres A, Malloy KE, Roy-Choudhury G, Kim J, Daadi MM Tags: Methods Mol Biol Source Type: research

Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach.
Abstract The directed differentiation of pluripotent stem cells to a desired lineage often involves complex and lengthy protocols. In order to study the requirements for differentiation in a systematic way, we present here methodology for an iterative approach using combinations of small molecules and biological factors. The factors are used in a cyclical process in which the best combination of factors and concentrations is selected in one round of testing, followed by a modification of the combination and subsequent rounds. While this may produce the desired differentiation in the cell population under study, it...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Farnsworth SL, Qiu Z, Mishra A, Hornsby PJ Tags: Methods Mol Biol Source Type: research

Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells.
Abstract Neural stem cells (NSCs) are multipotent and self-renewing precursor cells that give rise to all cell types of the central nervous system (CNS). They can be used for modeling CNS in vitro, for developmental studies and for cell replacement therapies. NSCs can be derived from pluripotent stem cells through differentiation using specific growth factors. Nonhuman primates (NHP) are critical preclinical models for translational research. Induced pluripotent stem cells (iPSCs) can be generated from NHP for the purposes of allogenic or autologous cell replacement studies. Here, we describe the derivation of NSC...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Hong H, Roy-Choudhury G, Kim J, Daadi MM Tags: Methods Mol Biol Source Type: research

Lentiviral Infection of Mouse Bone Marrow Cells for Hematopoietic Stem Cell Transplantation.
Abstract Lentiviral vectors are increasingly used as efficient gene transfer tools in the experimental and clinical gene therapy treatment of acquired and inherited genetic diseases. Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal, as well as multi-lineage differentiation and maintenance of the lymphohematopoietic system throughout life. As such, HSC transplantation (HSCT) has proven to be a powerful therapeutic modality for the treatment of both malignant and nonmalignant disorders. Transduction of lentiviral vectors into HSCs may offer long-term stable expression of a therapeut...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Chen C, Guderyon MJ, Ge G, Clark RA, Li S Tags: Methods Mol Biol Source Type: research

Central and Peripheral Secondary Cell Death Processes after Transient Global Ischemia in Nonhuman Primate Cerebellum and Heart.
Abstract Cerebral ischemia and its pathological sequelae are responsible for severe neurological deficits generally attributed to the neural death within the infarcted tissue and adjacent regions. Distal brain regions, and even peripheral organs, may be subject to more subtle consequences of the primary ischemic event which can initiate parallel disease processes and promote comorbid symptomology. In order to characterize the susceptibility of cerebellar brain regions and the heart to transient global ischemia (TGI) in nonhuman primates (NHP), brain and heart tissues were harvested 6 months post-TGI injury. I...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Lee JY, Lin R, Nguyen H, Russo E, Liska MG, Lippert T, Kaneko Y, Borlongan CV Tags: Methods Mol Biol Source Type: research

Method for Stimulation of Hippocampal Neurogenesis by Transient Microneedle Insertion.
Abstract Brief stereotaxic insertion and removal of a microneedle into the hippocampus of mice result in stimulation of hippocampal neurogenesis. This approach has been previously applied to a mouse model of Alzheimer's disease (Song et al., Cell Transplant 25:1853-1861, 2016). Further studies of fundamental cellular mechanisms of the brain's response to micro-injury will be useful for investigation of potential neuroprotective and deleterious effects of targeted microlesions and deep brain stimulation in neurodegenerative diseases. PMID: 30656634 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Song S, Kong X, Sanchez-Ramos J Tags: Methods Mol Biol Source Type: research

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.
Abstract Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based ...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Wickenhauser C, Bethmann D, Feng Z, Jensen SM, Ballesteros-Merino C, Massa C, Steven A, Bauer M, Kaatzsch P, Pazaitis N, Toma G, Bifulco CB, Fox BA, Seliger B Tags: Methods Mol Biol Source Type: research

Systems-Level Immune Monitoring by Mass Cytometry.
Abstract As therapies involving the modulation, stimulation, and deliberate excitation of the immune system are becoming routine, better methods for monitoring immune responses in human patients are needed. Mass cytometry allows for detailed profiling of all immune cell populations and their functional responses using a simple blood sample. When combined with appropriate computational analyses, the resolution for distinguishing desired responses from unproductive or even adverse reactions to immunotherapeutic interventions increases. Here we describe a core experimental and computational framework for global, syst...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Lakshmikanth T, Brodin P Tags: Methods Mol Biol Source Type: research

High-Throughput, Fast, and Sensitive Immunopeptidomics Sample Processing for Mass Spectrometry.
Abstract Comprehensive knowledge of the HLA class I and class II peptides presented to T cells is crucial for designing innovative therapeutics against cancer and other diseases. So far, methodologies for recovery of HLA class I and II peptides for subsequent mass spectrometry-based analysis have been a major limitation. In this chapter we describe a detailed protocol for a high-throughput, reproducible, and sensitive immunoaffinity-purification of HLA-I and HLA-II peptides from up to 96 samples in a plate format, suitable for tissue samples and cell lines. Our methodology reduces sample handling, has a competitiv...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Marino F, Chong C, Michaux J, Bassani-Sternberg M Tags: Methods Mol Biol Source Type: research

Expansion of Tumor-Infiltrating Lymphocytes from Melanoma Tumors.
vgren T Abstract Autologous, in vitro-expanded tumor-infiltrating lymphocytes (TIL) have been successfully used for treatment of melanoma patients. Expansion of TIL from tumors is usually performed in two steps. The details of the procedure differ between different laboratories, but the general concept remains the same. In the first step, small fragments of a tumor are placed in culture medium containing one or more T cell stimulating growth factors. Here the most common protocol using interleukin (IL)-2 is described. The length of the first step is flexible to allow generation of enough cell to start the second s...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Wickström S, Lövgren T Tags: Methods Mol Biol Source Type: research

Establishment of Two Dimensional (2D) and Three-Dimensional (3D) Melanoma Primary Cultures as a Tool for In Vitro Drug Resistance Studies.
LM, Rodríguez JA Abstract Characteristics of melanoma cells have been deciphered by studies carried out in two dimensional cell cultures growing as adherent monolayers on the bottom of plastic flasks. Melanoma cells can be cultured with a considerable degree of success, and, depending on the further use of the cells obtained in the culture, methodologies have to be adjusted to obtain reliable results. Although there are many melanoma continuous cell lines, in vitro 2D and 3D melanoma primary cell culture may be a more useful model to investigate interactions between cancer cells and immune system, as well a...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Cruz Rodríguez N, Lineros J, Rodríguez CS, Martínez LM, Rodríguez JA Tags: Methods Mol Biol Source Type: research

Assessment of Antitumor T-Cell Responses by Flow Cytometry After Coculture of Tumor Cells with Autologous Tumor-Infiltrating Lymphocytes.
tilde;a Y Abstract As a consequence of the ever-increasing use of immunotherapy for the treatment of cancer, interest in the direct interaction between T cells and tumors has surged tremendously. In vitro coculture of tumor cells with autologous tumor-infiltrating lymphocytes (TIL) is a highly physiological and clinically relevant model to study functional T-cell responses that result from the array of activatory and inhibitory signals that naturally occur on the patient's own tumor cells. This chapter describes a detailed protocol to set up a tumor-TIL coculture and assess ensuing functional T-cell responses, in ...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Melief J, Wickström S, Kiessling R, Pico de Coaña Y Tags: Methods Mol Biol Source Type: research

Expansion and Determination of Antigen-Reactive T Cells by Flow Cytometry.
Abstract The detection of antigen-reactive T cells has shown great utility for patient clinical monitoring. However, many of the methods commonly used to detect these cells face major limitations, like the predetermination of the given HLA type. The herein described protocol provides a means of overcoming many of the obstacles associated with the use of multimers and other common approaches in this field. In order to be able to detect rare cells which are below the detection limit of direct ex vivo measurement, in the present protocol, antigen-reactive T cells are first expanded in vitro using libraries of overlap...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Martens A, Pawelec G, Shipp C Tags: Methods Mol Biol Source Type: research

Assessing Canonical and Adaptive Natural Killer Cell Function in Suppression Assays In Vitro.
Abstract The in vitro culture system outlined in this chapter allows for standardized protocols to examine canonical and adaptive natural killer (NK) cell responses while interacting with immune suppressor cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). These interactions pathologically occur during tumorigenesis and tumor progression. Strategies to study the effects of ex vivo purified immune suppressor cells from cancer patients on the function of NK cell antitumor activity will help to understand suppressive mechanisms to improve immunotherapy. Immune checkpoint inhibitors h...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Sarhan D, Miller JS Tags: Methods Mol Biol Source Type: research

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay.
ña Y Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, th...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: van der Haar Àvila I, Marmol P, Kiessling R, Pico de Coaña Y Tags: Methods Mol Biol Source Type: research

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Flow Cytometry.
ña Y Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. I...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: van der Haar Àvila I, Marmol P, Cany J, Kiessling R, Pico de Coaña Y Tags: Methods Mol Biol Source Type: research

Evaluation of Breast Cancer and Melanoma Metastasis in Syngeneic Mouse Models.
Abstract Syngeneic mouse models enable us to study the interaction of the immune system with tumor cells during tumor development and metastasis. It further allows us to evaluate the efficacy of immunotherapies on the different stages of tumor growth and the metastatic process. Here we describe two syngeneic mouse models, a murine mammary carcinoma and a murine melanoma model, that are used to study metastasis. Metastases occur spontaneously in the murine mammary carcinoma. The presence and number of foci are evaluated by culturing the lung tissue in a colony formation assay. For the murine melanoma model, tumor c...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Witt K, Lundqvist A Tags: Methods Mol Biol Source Type: research

Establishment of Melanoma Tumor Xenograft Using Single Cell Line Suspension and Co-injection of Patient-Derived T Cells in Immune-Deficient NSG Mice.
We describe how this can be accomplished with injection of single cell suspension of melanoma tumor cells into immune-deficient NOD-scid IL2Rγnull (NSG) mice. We also describe how tumor cells and immune cells can be co-injected, using Winn assay, and the possibility to use that method to study immune therapies for cancer. PMID: 30666609 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Gunnarsdóttir FB, Kiessling R, Pico de Coaña Y Tags: Methods Mol Biol Source Type: research