1Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535
Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.
Published May 21, 2014. Keywords: Cellular Biology, Stress granule (SG), G3BP, primary cells, neurons
1Gene Expression Division, Bio-Rad Laboratories, Inc.
This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.
Published January 7, 2010. Keywords: Cellular Biology, Primary cell electroporation, MEF, Bio-Rad, Gene Pulser MXcell, transfection, GFP
1Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University
Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.
Published January 20, 2014. Keywords: Cellular Biology, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
1School of Biosciences, University of Birmingham
The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.
Published September 21, 2010. Keywords: Cellular Biology, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
1VECT-HORUS SAS, 2Aix-Marseille Université, CNRS, NICN UMR 7259
The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.…
Published June 28, 2014. Keywords: Medicine, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital
This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.
Published January 18, 2015. Keywords: Medicine, Biopsy, skeletal muscle, skin, tissue dissociation, myoblast purification, myoblast immortalization, cell freezing
1Department of Pathology, University of Virginia, 2Department of Obstetrics & Gynecology, University of Virginia
Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.
Published May 23, 2014. Keywords: Medicine, uterus, endometrium, endometrial stroma, (primary) cell culture, surgical blade, trypsin, tissue procurement, spontaneous decidualization
1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine
This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.
Published June 24, 2014. Keywords: Cellular Biology, live cell imaging, cardiomyocyte, primary cell culture, adenovirus, lentivirus, confocal spinning disk microscopy
1Department of Biological Sciences, Purdue University, 2Department of Oncology, University of Alberta, 3Cross Cancer Institute
In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.
Published March 8, 2014. Keywords: Medicine, extracellular matrix, 3D culture, bone marrow, hematological malignancies, primary cell culture, tumor microenvironment
1Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2College of Medicine, University of Illinois at Urbana-Champaign, 3Provena Covenant Medical Centre, 4Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign
A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.
Published June 4, 2015. Keywords: Bioengineering, Primary human colon tumor cells, Soft Elastic Substrates, Traction force Microscopy, Mechanobiology, Immunofluorescence Microscopy, Cell mechanics
1Department of Medicine, Keck School of Medicine, University of Southern California
We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions. …
Published January 18, 2015. Keywords: Cellular Biology, Cellular biology, mouse, human, esophagus, mesenchymal stromal cells, myofibroblasts, primary cells
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
Published March 13, 2013. Keywords: Cancer Biology, Medicine, Stem Cell Biology, Cellular Biology, Molecular Biology, Biomedical Engineering, Genetics, Oncology, Primary cell culture, melanoma, MACS, cancer stem cells, CD133, cancer, prostate cancer cells, melanoma, stem cells, cell culture, personalized treatment
1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 2Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 3Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 4Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, 5Biological and Biomedical Sciences Program, University of North Carolina School of Medicine, 6Department of Radiation Oncology, Emory University School of Medicine, 7Department of Neurology, Neurosciences Center, University of North Carolina School of Medicine
Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.
Published August 12, 2014. Keywords: Neuroscience, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
JoVE Developmental Biology
1Centre of Human and Aerospace Physiological Sciences, King's College London, 2Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute
The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.
Published January 12, 2015. Keywords: Developmental Biology, Stem cell Biology, Tissue Engineering, Stem Cells, Satellite Cells, Skeletal Muscle, Adipocytes, Myogenic Cells, Myoblasts, Fibroblasts, Magnetic Activated Cell Sorting, Image Analysis
JoVE Immunology and Infection
1Department of Immunology and Microbiology, Rush University Medical Center
Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.
Published March 14, 2011. Keywords: Immunology, innate immunity, HIV-1, natural killer cell, cytolytic assay, degranulation assay, primary lymphocytes
1Medical Biotechnology, Technische Universität Berlin, 2TissUse GmbH, 3Fraunhofer IWS
Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.
Published April 28, 2015. Keywords: Bioengineering, Multi-organ chip, human-on-a-chip, body-on-a-chip, organs-on-a-chip, microphysiological systems, organoids, tissue engineering, in vitro substance testing, toxicity test, liver, skin, vasculature
1Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota, 2Department of Obstetrics and Gynecology, Maricopa Medical Center and St Josephs Hospital and Medical Center, 3Masonic Cancer Center, University of Minnesota
This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.
Published February 4, 2014. Keywords: Medicine, Neoplasms, Ovarian Cancer, Primary cell lines, Clinical Specimens, Downstream Applications, Targeted Therapies, Epithelial Cultures
1Department of Materials Science and Engineering, University of Sheffield, 2Department of Oncology and Insigneo Institute for in silico Medicine, University of Sheffield, 3Department of Histopathology, Sheffield Teaching Hospitals NHS Foundation Trust
This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.
Published May 18, 2015. Keywords: Bioengineering, esophagus, epithelium, tissue engineering, 3D construct, esophageal cancer, Barrett’s Metaplasia
1Medicine, Faculty of Health Sciences, McMaster University
Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.
Published March 26, 2010. Keywords: Medicine, Human bronchus, epithelium, primary culture, permeable support, cilia
1Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, 2Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca' Granda
Ospedale Maggiore Policlinico, 3Renal Physiopathology Laboratory, Department of Medical, Surgical and Health Sciences, University of Trieste
Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.
Published May 13, 2014. Keywords: Cellular Biology, cell biology, cell culture, bone, retinoic acid, primary osteoblasts, osteocytes, cell differentiation, mouse calvaria, sclerostin, fibroblast growth factor 23, microscopy, immunostaining
JoVE Developmental Biology
1Biomedical Sciences Program, Midwestern University, 2Department of Microbiology & Immunology, Midwestern University
Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.
Published February 25, 2015. Keywords: Developmental Biology, Collective cell migration, reepithelization, zebrafish, keratocyte, epithelial to mesenchymal transition, cell-cell adhesion, cutaneous wound healing
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine
The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.
Published February 16, 2015. Keywords: Cellular Biology, Anastasis, apoptosis, apoptotic bodies, caspase, cell death, cell shrinkage, cell suicide, cytochrome c, DNA damage, genetic alterations, mitochondrial outer membrane permeabilization (MOMP), programmed cell death, reversal of apoptosis
JoVE Immunology and Infection
1Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia, 2Department of Biology, University of Pisa, 3Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia
Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.
Published March 28, 2014. Keywords: Immunology, Flow cytometry, blood leukocytes, microglia, Nanoparticles, internalization, Fluorescence, cell purification
1Australian Centre for Blood Diseases, Monash University
Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.
Published November 12, 2013. Keywords: Bioengineering, Blood-brain barrier, model, cell culture, astrocytes, brain endothelial cells, insert, membranes
JoVE Immunology and Infection
1Research Group Immune Regulation, Helmholtz Centre for Infection Research, 2Research Group Infection Immunology, Institute of Medical Microbiology, Otto-von-Guericke University, 3Department of Experimental Immunology, Helmholtz Centre for Infection Research
We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.
Published December 26, 2012. Keywords: Immunology, Cellular Biology, Molecular Biology, Infection, Infectious Diseases, Microbiology, alveolar type II epithelial cells, mouse, respiratory tract, lung, cell sorting, flow cytometry, influenza, autoimmunity
1Department of Surgery, UCLA, 2Department of Pathology, UC Irvine
The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.
Published October 12, 2013. Keywords: Cellular Biology, Myofibroblasts, Mesenchymal Stromal Cells, Gastrointestinal Tract, stroma, colon, primary cells
1Program in Epithelial Biology, Stanford University School of Medicine
Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.
Published February 28, 2013. Keywords: Genetics, Tissue Engineering, Medicine, Biomedical Engineering, Cellular Biology, Surgery, Epithelial Biology, regeneration, chamber, hair, follicle, dermis, dermal cells, keratinocyte, graft, epithelial, cell culture, lentivirus, knockdown, shRNA-mediated knockdown, overexpression, mice, transgenic mice, animal model
1Murdoch Childrens Research Institute, The Royal Childrens Hospital, 2Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton
The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.
Published May 1, 2015. Keywords: Medicine, lymphatic endothelial cell, lymphatic malformation, flow cytometric sorting, cell culture, cell surface markers
1Stem Cells & Cancer Group, Molecular Pathology Program, Spanish National Cancer Research Center, 2Institute for Research in Biomedicine (IRB Barcelona), 3Center for Stem Cells in Cancer & Ageing, Barts Cancer Institute, Queen Mary University of London
Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.
Published June 20, 2015. Keywords: Medicine, Pancreatic ductal adenocarcinoma, cancer stem cells, spheres, metformin (met), metabolism
1INSERM U1052, Centre de Recherche en Cancérologie de Lyon, 2CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, 3Université de Lyon, 4Université Lyon 1, 5Centre Léon Bérard
In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.
Published August 13, 2013. Keywords: Cancer Biology, Cellular Biology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Surgery, Oncology, Pancreas, Exocrine, Cells, Cultured, Mice, Primary Cell Culture, Exocrine pancreas, Cell culture, Primary acinar cells, Mouse, pancreatic cancer, cancer, tumor, tissue, animal model
1Anchored Signalling, Max-Delbrück-Center for Molecular Medicine, 2Leibniz Institute for Molecular Pharmacology (FMP), 3Charité University Medicine Berlin
Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.
Published June 21, 2013. Keywords: Cellular Biology, Bioengineering, Genetics, Molecular Biology, Biochemistry, Biomedical Engineering, Medicine, Pharmacology, Intercellular Signaling Peptides and Proteins, Exocytosis, Signal Transduction, Second Messenger Systems, Calcium Signaling, Cardiovascular Diseases, Hormones, Hormone Substitutes, and Hormone Antagonists, Life Sciences (General), water reabsorption, kidney, principal cells, vasopressin, cyclic AMP, aquaporin, animal model, cell culture
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
Published May 7, 2013. Keywords: Cellular Biology, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
1Division of Molecular Oncology, IRCCS, San Raffaele Scientific Institute, 2Department of Haemato-Oncology, King's College London, 3IFOM, FIRC Institute of Molecular Oncology, 4Università Vita-Salute San Raffaele
Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.…
Published October 19, 2014. Keywords: Medicine, Lymphocytes, Chronic Lymphocytic Leukemia, 2D Electrophoresis, Mass Spectrometry, Cytoskeleton, Migration
1Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg
Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.
Published August 6, 2013. Keywords: Cancer Biology, Biomedical Engineering, Bioengineering, Medicine, Anatomy, Physiology, Molecular Biology, Cellular Biology, Tissue Engineering, Tumor Cells, Cultured, Biotechnology, Culture Techniques, Cell Engineering, Cellular Microenvironment, Equipment and Supplies, Decellularization, BioVaSc, primary cell isolation, tumor test system, dynamic culture conditions, bioreactor, 3D in vitro models, cell culture
1Randall and Cardiovascular Divisions, King’s College London, 2Division of Cardiology, School of Medicine, University of California San Diego
Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.…
Published September 6, 2013. Keywords: Cellular Biology, Biomedical Engineering, Bioengineering, Molecular Biology, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Disease Models, Animal, Models, Cardiovascular, Cell Biology, neonatal mouse, cardiomyocytes, isolation, culture, primary cells, NMC, heart cells, animal model
1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International
We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.
Published May 17, 2012. Keywords: Neuroscience, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
JoVE Immunology and Infection
1Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud university medical center
Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.
Published December 10, 2013. Keywords: Immunology, Blood Cells, Respiratory Syncytial Virus, Human, Respiratory Tract Infections, Paramyxoviridae Infections, Models, Immunological, Immunity, HRSV culture, purification, quantification, PBMC isolation, stimulation, inflammatory pathways
1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute
We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.
Published February 28, 2011. Keywords: Development Biology, Primary cell culture, Drosophila embryo, RNAi, immuno-histochemistry
JoVE Immunology and Infection
1Biological Sciences Division, Pacific Northwest National Laboratory, 2Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, 3Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, 4Center for Bioproducts and Bioenergy, Washington State University
Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. …
Published January 19, 2015. Keywords: Immunology, electroporation, protein, transfection, expression, localization, confocal microscopy, Salmonella, effector
JoVE Immunology and Infection
1Department of Cardiology, University of Heidelberg
Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.
Published June 12, 2013. Keywords: Immunology, Infection, Medicine, Cellular Biology, Molecular Biology, Inflammation, Monocyte-Macrophage Precursor Cells, Myeloid Cells, Immune System, Macrophages, Mononuclear Phagocyte System, Cells, in vitro model, human, cell culture, differentiation, polarization
JoVE Immunology and Infection
1Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, 2Department of Medical Biology, University of Melbourne
Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.
Published July 17, 2014. Keywords: Immunology, Parasitic Diseases, malaria, Plasmodium falciparum, hemozoin, antibody, Fc Receptor, opsonization, merozoite, phagocytosis, THP-1
1Department for Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 2Translational Center Würzburg, Regenerative Therapies in Oncology and Musculoskelettal Disease, Würzburg Branch of the Fraunhofer-Institute Interfacial Engineering and Biotechnology, IGB
The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.
Published February 26, 2015. Keywords: Bioengineering, Tissue engineering, 3D in vitro models, test system, alternative to animal testing, full thickness, skin equivalent, skin injury, wound model, automation, wounding device
JoVE Immunology and Infection
1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille
Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.…
Published January 17, 2014. Keywords: Infection, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
1Department of Cell Biology, Harvard Medical School
The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.
Published February 17, 2012. Keywords: Medicine, Ovarian Cancer, Metastasis, In vitro Model, Mesothelial, Spheroid
1UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco, 2Department of Biology, University of Copenhagen, Denmark, 3National Institute of Nutrition and Seafood Research, Bergen, Norway
Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.
Published March 28, 2013. Keywords: Cellular Biology, Medicine, Anatomy, Physiology, Molecular Biology, Surgery, Adipose Tissue, Adipocytes, Transcription Factors, Cell Differentiation, Obesity, Diabetes, brown adipose tissue, beige/brite cells, primary adipocytes, stromal-vascular fraction, differentiation, uncoupling protein 1, rosiglitazone, differentiation, cells, isolation, fat, animal model
JoVE Immunology and Infection
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
Published July 16, 2012. Keywords: Virology, Immunology, Molecular Biology, Genetics, cell fusion, HCV, restriction factor, heterokaryon, mouse, species-specificity
1McAllister Heart Institute, University of North Carolina at Chapel Hill
Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.
Published July 20, 2013. Keywords: Cellular Biology, Molecular Biology, Biomedical Engineering, Bioengineering, Medicine, Cardiology, Cells, Cultured, Embryo, Mammalian, Endothelium, Vascular, Heart, primary cells, cell isolation, endothelial cells, cell, cell culture
1Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center
The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.
Published May 20, 2015. Keywords: Medicine, Tumor, breast, lung, primary, MEF, embryo, fibroblasts, cancer, cell, mouse
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).
Published August 21, 2011. Keywords: Neuroscience, Oligodendrocyte, myelination, in vitro, dorsal root ganglion neuron, co-culture, primary cells, mouse, neuroscience
1Department of Molecular & Integrative Physiology, University of Michigan, 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan
A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.
Published May 17, 2013. Keywords: Cancer Biology, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Oncology, Surgery, Organoids, Tumor Cells, Cultured Colonic Neoplasms, Primary Cell Culture, Colon tumor, chelation, collagenase, matrigel, organoid, EGF, colon cancer, cancer, tumor, cell, isolation, immunohistochemistry, mouse, animal model