Cell Cycle:
The complex series of phenomena, occurring between the end of one Cell division and the end of the next, by which cellular material is duplicated and then divided between two daughter cells. The cell cycle includes Interphase, which includes G0 phase; G1 phase; S Phase; and G2 phase, and Cell division phase.
JoVE General
Analysis of Cell Cycle Position in Mammalian Cells
1Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 2London Regional Cancer Program, Children's Health Research Institute, and Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario
Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.
JoVE General
Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Department of Cell and Molecular Biology, Karolinska Institutet
FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.
JoVE General
Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry
Faculty of Pharmaceutical Sciences, University of British Columbia
Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.
JoVE General
Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay
A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.
JoVE General
Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos
Department of Biological Sciences, University of North Texas
Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.
JoVE General
Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
Department of Oncology, University of Wisconsin - Madison
A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.
JoVE General
Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast
Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University
Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.
JoVE General
Preparation of Drosophila S2 cells for Light Microscopy
Department of Cell Biology and Anatomy, University of Arizona (UOA)
Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.
JoVE General
Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System
Laboratory of Molecular Gerontology, National Institute on Aging, NIH
Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.
JoVE General
Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue
We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.
JoVE General
Measuring Replicative Life Span in the Budding Yeast
1Department of Biochemistry, University of Washington, 2Department of Pathology, University of Washington
In this article we present a general protocol for measuring the replicative life span of yeast mother cells.
JoVE General
Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano
A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.
JoVE General
Purification of Progenitors from Skeletal Muscle
The Biomedical Research Centre, University of British Columbia
Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.
JoVE General
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
JoVE General
Growth Assays to Assess Polyglutamine Toxicity in Yeast
Boston Biomedical Research Institute
This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.
JoVE General
Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells
1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver
In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.
JoVE General
Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
Department of Biology, University of Rochester
This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.
JoVE Immunology and Infection
Generation of Organotypic Raft Cultures from Primary Human Keratinocytes
1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill, 2Lineberger Cancer Center, University of North Carolina-Chapel Hill
An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.
JoVE General
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
JoVE General
Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels
Department of Pharmacology, University of Pennsylvania
The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.
JoVE General
Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model
Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.
JoVE Bioengineering
A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School
We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.
JoVE Immunology and Infection
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
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.
JoVE Immunology and Infection
Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood
1Division of Pediatrics, U.T. MD Anderson Cancer Center, 2Department of Stem Cell Transplantation and Cellular Therapy, U.T. MD Anderson Cancer Center
T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.
JoVE General
MISSION esiRNA for RNAi Screening in Mammalian Cells
Max Planck Institute of Molecular Cell Biology and Genetics
Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.
JoVE Neuroscience
Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae
Epigenetics and Progenitor Cells Keystone, Fox Chase Cancer Center
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
JoVE Neuroscience
Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Department of Cell and Developmental Biology, Vanderbilt University
This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.
JoVE General
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta
An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.
JoVE General
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.
JoVE Neuroscience
Single-cell Profiling of Developing and Mature Retinal Neurons
Department of Genetics, Development and Cell Biology, Neuroscience Program, Iowa State University
A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.
JoVE Immunology and Infection
Reverse Genetics Mediated Recovery of Infectious Murine Norovirus
Section of Virology, Imperial College London
Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.
JoVE General
Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg
Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.
JoVE Clinical and Translational Medicine
Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University
Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.
JoVE General
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
JoVE General
Title Cell Encapsulation by Droplets
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's, Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 3Brigham and Women's Hospital, Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital
JoVE General
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
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.
JoVE Immunology and Infection
An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells
Infectious Disease Research Center, CHUL (CHUQ), Quebec City, Quebec, Canada
We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.
JoVE General
Primary Cell Cultures from Drosophila Gastrula Embryos
1Department of Genetics, Harvard Medical School, 2 , Howard 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.
JoVE General
Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification
1Department of Biochemistry, Microbiology and Immunology, Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3CIHR Program in Neurodegenerative Lipidomics, University of Ottawa, 4Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism
Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.
JoVE General
Generation of Human CD40-activated B cells
In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.
JoVE Immunology and Infection
Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
JoVE General
Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.
JoVE Clinical and Translational Medicine
Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)
1Department of Neurosurgery, The University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.
JoVE General
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Department of Biological Sciences, The University of Memphis
Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.
JoVE Applied Physics
Construction and Testing of Coin Cells of Lithium Ion Batteries
1School of Materials Science and Engineering, Clemson University, 2Center for Optical Materials Science and Engineering Technologies, Clemson University
A protocol to construct and test coin cells of lithium ion batteries is described. The specific procedures of making a working electrode, preparing a counter electrode, assembling a cell inside a glovebox and testing the cell are presented.
JoVE General
Chromatin Immunoprecipitation from Human Embryonic Stem Cells
Department of Biochemistry, University of California - Riverside
The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.
JoVE General
Using Laser Tweezers For Manipulating Isolated Neurons In Vitro
This video describes the manipulation of cultured neurons using laser tweezers in vitro.
JoVE Immunology and Infection
Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Department of Immunology, University of Toronto
Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
JoVE Immunology and Infection
Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
1Immunology, University of Toronto, 2Arthritis and Immune Disorder Research Centre, University Health Network (UHN)
This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.
JoVE Immunology and Infection
Recombinant Retroviral Production and Infection of B Cells
1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 2Herbert Irving Comprehensive Cancer Center, Columbia University
An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.
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