1Department of Oncological Sciences, University of Utah
Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.
Published December 20, 2013. Keywords: Developmental Biology, zebrafish, embryo, apoptosis, Caspase 3, Immunofluorescence, whole-mount, cell death
1Department of Molecular and Cellular Biology, University of Guelph
Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.
Published January 13, 2012. Keywords: Neuroscience, myelin basic protein, apoptosis, neuroprotection, caspase-3, live-cell imaging, glia, oligodendrocytes
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
1Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System, 2Department of Food Science and Nutrition, University of Minnesota, 3Department of Integrative Biology and Physiology, University of Minnesota, 4Department of Medicine, University of Minnesota Medical School, University of Minnesota
Multiplex assays can provide beneficial information for basic cellular mechanisms and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.…
Published April 16, 2014. Keywords: Neuroscience, apoptosis, obesity, caspase, resazurin, DEVD, palmitic acid, hypothalamic model
JoVE Immunology and Infection
1Department Biologie, Friedrich-Alexander-Universität, 2Institut für Molekulare Pathogenese, Friedrich-Loeffler-Institut, 3Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen
The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.
Published June 25, 2015. Keywords: Infection, Apoptosis, Bax, Caspase 3, Coxiella burnetii, Doxycycline, Effector protein, Inducible expression, stable cell line, Tet system, Type IV Secretion System
1Neurodevelopment Group, School of Biosciences, University of Birmingham
An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.…
Published March 28, 2013. Keywords: Neurobiology, Developmental Biology, Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Bioengineering, Central Nervous System, Neuroglia, Drosophila, fruit fly, animal models, Wounds and Injuries, Cell Physiological Phenomena, Genetic Phenomena, injury, repair, regeneration, central nervous system, ventral nerve cord, larva, live imaging, cell counting, Repo, GS2, glia, neurons, nerves, CNS, animal model
1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.…
Published January 4, 2012. Keywords: Bioengineering, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
1Pediatric Airway Research Center, Department of Pediatrics, University of Colorado, 2Department of Chemical and Biological Engineering, Colorado School of Mines
This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.…
Published May 8, 2014. Keywords: Bioengineering, air-liquid interface, chlorine exposure, toxic inhaled chemicals, Transepithelial Electrical Resistance, Immunocytochemistry
1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University
Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.
Published November 28, 2012. Keywords: Medicine, Neuroscience, Molecular Biology, Ophthalmology, Immunology, UVR-B, lens, cataract, qRT-PCR, PCR, immunohistochemistry, rat restrainer, animal model
1Department of Chemistry, Tunghai University, 2Department of Biochemistry, University of California, Riverside
MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.
Published November 26, 2011. Keywords: Biochemistry, Deuterium, H/D exchange, Mass Spectrometry, Pak2, Caspase 3, MALDI-TOF
1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 2Department of Rheumatology, Tufts Medical Center
A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.
Published January 31, 2012. Keywords: Cellular Biology, Chondrocytes, articular, human, synovial fluid, alginate bead, 3D culture
1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed “tumor explant method”. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide. …
Published July 29, 2011. Keywords: Medicine, Glioblastoma multiforme, glioma, temozolomide, therapeutics, drug design
1Cell Biology, University of Bielefeld, 2Molecular Neurobiology, University of Bielefeld
Methods for the manipulation and analysis of NF-κB-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.…
Published February 13, 2014. Keywords: Neuroscience, NF-κB, hippocampus, Adult neurogenesis, spatial pattern separation-Barnes maze, dentate gyrus, p65 knock-out mice
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
JoVE Immunology and Infection
1Department of Neurology, University of Münster, 2Germany and Interdisciplinary Center for Clinical Research (IZKF) Münster, 3Institute of Physiology I - Neuropathophysiology I, University of Münster
To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.…
Published February 5, 2015. Keywords: Immunology, acute brain slices, multiple sclerosis, MS, ex vivo model, autoimmunity, neuroinflammation, central nervous system
1Division of Neuropathology, Department of Pathology, Pathobiology Graduate Program, Johns Hopkins School of Medicine
This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.
Published December 16, 2014. Keywords: Physiology, TUNEL, fluorescence, skeletal muscle, DNA damage, image analysis, histology, SMA, motor neuron disease
1Institute of Reconstructive Neurobiology, University of Bonn
A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.
Published January 31, 2012. Keywords: Neuroscience, Developmental Biology, organotypic slice culture, midbrain, mouse, time-lapse imaging, neurons
1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey
We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.
Published August 14, 2012. Keywords: Cancer Biology, Medicine, Neuroscience, Cell Culture, Tissue Engineering, 3D model, multicellular spheroids, therapeutic drugs, neuroendocrine tumor cell lines, agarose overlay platform, paraffin embedding
JoVE Immunology and Infection
1Department of Medical Microbiology and Immunology, University of Alberta
We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.
Published October 8, 2012. Keywords: Immunology, Medicine, Cellular Biology, Anatomy, Physiology, Thymus, T cell, negative selection, positive selection, autoimmunity, flow cytometry
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences
The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.…
Published January 7, 2013. Keywords: Cellular Biology, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
1Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 2Department of Pathology, University of Michigan Comprehensive Cancer Center
Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.
Published April 25, 2014. Keywords: Medicine, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
1Cellular and Molecular Medicine Program, Boston Children’s Hospital and Harvard Medical School, 2Department of Medicine, University of Fribourg, 3Centre Médical Universitaire, University of Geneva
We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.
Published June 10, 2015. Keywords: Biochemistry, Granzyme, immune serine protease, cell-mediated cytotoxicity, recombinant protein production, mammalian expression system, protein purification
1Department of Anatomy and Cell Biology and the Rappaport Institute for Research in the Medical Sciences, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology
Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.
Published August 18, 2013. Keywords: Developmental Biology, Cellular Biology, Molecular Biology, Genetics, Bioengineering, Drosophila, Immunity, Innate, Phagocytosis, Apoptosis, Genes, Developmental, Cell Biology, biology (general), genetics (animal and plant), life sciences, embryo, glia, fruit fly, animal model
1Department of Chemical Engineering, University Of Massachusetts Amherst
We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.
Published November 20, 2011. Keywords: Bioengineering, Microfluidic Device, Tumor Microenvironment, Hanging Drop Spheroids, Apoptosis, Drug Penetration
1Department of Chemistry and Biochemistry, University of Windsor, 2Chemistry Department and Centre for Biotechnology, Brock University
We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.…
Published May 30, 2012. Keywords: Cancer Biology, Medicine, Biochemistry, Breast adenocarcinoma, neuroblastoma, tamoxifen, combination therapy, apoptosis, autophagy
1Resuscitation Institute, Rosalind Franklin University of Medicine and Science
This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.…
Published April 26, 2015. Keywords: Medicine, Cardiopulmonary resuscitation, Hemodynamics, Myocardial ischemia, Rats, Reperfusion, Ventilation, Ventricular fibrillation, Ventricular function, Translational medical research
1Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Spemann Graduate School of Biology and Medicine and Faculty of Biology, University of Freiburg, 3School of Life Sciences, Keele University, 4Center for Biological Signaling Studies (BIOSS), University of Freiburg
We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.
Published December 18, 2014. Keywords: Neuroscience, CD markers, surface antigens, intracellular antigens, flow cytometry, neurons, glial cells, neural stem cells, fluorescence-activated cell sorting (FACS), CD24, CD54, CFSE
1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida
This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.…
Published May 23, 2012. Keywords: Medicine, Physiology, type 1 diabetes, histology, H&E, immunohistochemistry, insulin, beta-cells, glucagon, alpha-cells, pancreatic polypeptide, islet, pancreas, spleen, organ donor
1Dr. William M. Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Chicago Medical School, Rosalind Franklin University of Medicine and Science
Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.
Published February 28, 2013. Keywords: Medicine, Genetics, Genomics, Physiology, Anatomy, Biomedical Engineering, Metabolomics, Zebrafish, diabetes, metabolic memory, tissue regeneration, streptozocin, epigenetics, Danio rerio, animal model, diabetes mellitus, diabetes, drug discovery, hyperglycemia
1Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, 2Department of Chemistry, Rice University, 3Mechanical Engineering and Materials Science, Rice University
We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.
Published August 28, 2013. Keywords: Medicine, Electronics and Electrical Engineering, Life Sciences (General), Radiofrequency, Cancer, Nanoparticles, Hyperthermia, Gold
1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.
A combination of different techniques to maximize data collection from mouse tissue is presented.
Published March 17, 2011. Keywords: Neuroscience, lipophilic dye, in situ hybridization, immunohistochemistry, histology, neuronal tracing
1Department of Neurology, Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Germany
There is accumulating evidence, that ischemic preconditioning (PC) – a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.…
Published May 9, 2013. Keywords: Medicine, Neurobiology, Anatomy, Physiology, Neuroscience, Immunology, Surgery, stroke, cerebral ischemia, ischemic preconditioning, ischemic tolerance, IT, ischemic stroke, middle cerebral artery occlusion, MCAO, bilateral common carotid artery occlusion, BCCAO, brain, ischemia, occlusion, reperfusion, mice, animal model, surgical techniques
1Department of Ophthalmology, Inselspital, University of Bern, 2Department of Ophthalmology, University Hospital of Basel, 3Department of Biology, University of Fribourg
Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.…
Published October 20, 2014. Keywords: Cellular Biology, N-methyl-N-nitrosourea (MNU), retina, degeneration, photoreceptors, Müller cells, regeneration, zebrafish, visual function
1Keenan Research Centre of the LiKaShing Knowledge Institute, St Michaels Hospital, 2Department of Surgery, McMaster University
The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.
Published November 3, 2013. Keywords: Medicine, mouse, tibial nerve, gastronemius, soleus, atrophy, denervation, reinnervation, myofiber, transection
1Department of Surgery, Texas A&M University Health Science Center College of Medicine, 2Department of Surgery, Baylor Scott & White Health
Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.
Published May 7, 2015. Keywords: Medicine, Oxygen-glucose deprivation and reoxygenation, ischemia-reperfusion injury, blood-brain barrier, brain endothelial cells, tight junctions, immunofluorescence, f-actin staining
1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine
Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.
Published January 18, 2012. Keywords: Bioengineering, Extravillous trophoblasts, cytotrophoblast, invasion, matrix metalloproteinase, 3-D cell culture, RCCS, ECM, microcarriers
1Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado, 2Department of Anesthesiology, University of Colorado
The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.
Published March 3, 2014. Keywords: Medicine, Spinal cord injury, thoracic aorta, paraplegia, Ischemia, reperfusion, murine model
1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg
Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.
Published November 21, 2012. Keywords: Neuroscience, Medicine, Cellular Biology, Anatomy, Physiology, Developmental Biology, Schwann cell, migration, time-lapse, SCG, neurons, axons, mouse
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
Published July 16, 2011. Keywords: Cellular Biology, Human hematopoietic stem cells, erythropoiesis, red blood cells, lentivirus, knockdown, transcription factor