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Caspase 3: A short pro-domain caspase that plays an effector role in Apoptosis. It is activated by Initiator caspases such as Caspase 9. Isoforms of this protein exist due to multiple alternative splicing of its Messenger rna.
 JoVE Biology

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

1Department of Oncological Sciences, University of Utah


JoVE 51060

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.

 JoVE Neuroscience

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

1Department of Molecular and Cellular Biology, University of Guelph


JoVE 3422

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.

 JoVE Neuroscience

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

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


JoVE 51305

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.

 JoVE Neuroscience

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

1Neurodevelopment Group, School of Biosciences, University of Birmingham


JoVE 50306

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.

 JoVE Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University


JoVE 3612

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.

 JoVE Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

1Pediatric Airway Research Center, Department of Pediatrics, University of Colorado, 2Department of Chemical and Biological Engineering, Colorado School of Mines


JoVE 51539

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.

 JoVE Clinical and Translational Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University


JoVE 4016

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.

 JoVE Biology

Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

1Department of Chemistry, Tunghai University, 2Department of Biochemistry, University of California, Riverside


JoVE 3602

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

 JoVE Biology

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 2Department of Rheumatology, Tufts Medical Center


JoVE 3587

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.

 JoVE Clinical and Translational Medicine

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center


JoVE 2846

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.

 JoVE Neuroscience

Methods for the Modulation and Analysis of NF-κB-dependent Adult Neurogenesis

1Cell Biology, University of Bielefeld, 2Molecular Neurobiology, University of Bielefeld


JoVE 50870

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.

 JoVE Biology

Viability Assays for Cells in Culture

1Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University


JoVE 50645

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.

 JoVE Biology

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

1Division of Neuropathology, Department of Pathology, Pathobiology Graduate Program, Johns Hopkins School of Medicine


JoVE 52211

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.

 JoVE Neuroscience

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

1Institute of Reconstructive Neurobiology, University of Bonn


JoVE 3350

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.

 JoVE Clinical and Translational Medicine

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

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


JoVE 4218

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.

 JoVE Immunology and Infection

Examination of Thymic Positive and Negative Selection by Flow Cytometry

1Department of Medical Microbiology and Immunology, University of Alberta


JoVE 4269

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.

 JoVE Biology

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

1Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences


JoVE 4301

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.

 JoVE Clinical and Translational Medicine

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

1Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 2Department of Pathology, University of Michigan Comprehensive Cancer Center


JoVE 51311

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.

 JoVE Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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


JoVE 50151

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.

 JoVE Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

1Department of Chemical Engineering, University Of Massachusetts Amherst


JoVE 2425

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.

 JoVE Clinical and Translational Medicine

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

1Department of Chemistry and Biochemistry, University of Windsor, 2Chemistry Department and Centre for Biotechnology, Brock University


JoVE 3586

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.

 JoVE Neuroscience

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

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


JoVE 52241

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.

 JoVE Clinical and Translational Medicine

Staining Protocols for Human Pancreatic Islets

1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida


JoVE 4068

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.

 JoVE Clinical and Translational Medicine

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

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


JoVE 50232

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.

 JoVE Clinical and Translational Medicine

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

1Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, 2Department of Chemistry, Rice University, 3Mechanical Engineering and Materials Science, Rice University


JoVE 50480

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.

 JoVE Neuroscience

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.


JoVE 2451

A combination of different techniques to maximize data collection from mouse tissue is presented.

 JoVE Clinical and Translational Medicine

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

1Department of Neurology, Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Germany


JoVE 4387

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.

 JoVE Biology

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

1Department of Ophthalmology, Inselspital, University of Bern, 2Department of Ophthalmology, University Hospital of Basel, 3Department of Biology, University of Fribourg


JoVE 51909

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.

 JoVE Clinical and Translational Medicine

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

1Keenan Research Centre of the LiKaShing Knowledge Institute, St Michaels Hospital, 2Department of Surgery, McMaster University


JoVE 50657

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.  

 JoVE Bioengineering

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

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


JoVE 3367

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.

 JoVE Clinical and Translational Medicine

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

1Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado, 2Department of Anesthesiology, University of Colorado


JoVE 50910

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.

 JoVE Neuroscience

Analyzing Murine Schwann Cell Development Along Growing Axons

1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg


JoVE 50016

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

 JoVE Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa


JoVE 2813

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.

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