1Department of Cell and Developmental Biology, University of Pennsylvania, 2Biomedical Graduate Studies, University of Pennsylvania, 3Department of Animal Biology and Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, 4Department of Genetics, University of Pennsylvania, 5Department of Biology, University of Pennsylvania
The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.
Published October 9, 2013. Keywords: Cellular Biology, Developmental Biology, Spermatogenesis, STA-PUT, cell separation, Spermatogenesis, spermatids, spermatocytes, spermatogonia, sperm, velocity sedimentation
1Biochemistry and Molecular Biology, Colorado State University
A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.
Published September 10, 2013. Keywords: Cellular Biology, Chromosome Structures, Chromatin, Nucleosomes, Histones, Microscopy, Atomic Force (AFM), Biochemistry, Chromatin, Nucleosome, Nucleosomal Array, Histone, Analytical Ultracentrifugation, Sedimentation Velocity
1Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen
Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.
Published November 16, 2013. Keywords: Basic Protocols, FtsZ, protein polymerization, cell division, GTPase, sedimentation assay, light scattering
1Department of Developmental and Cell Biology, School of Biosciences, University of California, Irvine
This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.
Published April 27, 2012. Keywords: Developmental Biology, Drosophila, Kinesin, clarification, polymerization, sedimentation, microtubule
1National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science
The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.
Published November 5, 2009. Keywords: Basic Protocols, analytical ultracentrifugation, sedimentation velocity, sedimentation equilibrium, protein characterization, sedimentation coefficient
1Department of Chemical and Biological Engineering, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa
Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.
Published April 25, 2013. Keywords: Bioengineering, Biophysics, Chemical Engineering, Mechanical Engineering, Biomedical Engineering, Medicine, Anatomy, Physiology, Cellular Biology, Molecular Biology, Hematology, Blood Physiological Phenomena, Hemorheology, Hematocrit, flow characteristics, flow measurement, flow visualization, rheology, Red blood cells, cross correlation, micro blood flows, microfluidics, microhemorheology, microcirculation, velocimetry, visualization, imaging
JoVE Applied Physics
1Department of Physics, The University of Chicago, 2James Franck Institute, The University of Chicago, 3Department of Mechanical Engineering and Materials Science, Yale University
Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.
Published March 5, 2014. Keywords: Physics, fluid mechanics, fast camera, dense suspension, liquid metal, drop impact, splashing
1Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), 2Servicio de Citometría de Flujo y Clasificación Celular (SECIF), Instituto de Investigaciones Biológicas Clemente Estable, 3Sección Bioquímica, Facultad de Ciencias, Universidad de la República
A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.
Published August 4, 2013. Keywords: Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Cell Separation, Flow Cytometry, Cytological Techniques, Meiosis, Spermatogenesis, Cell Biology, Flow cytometry, FACS, testis, meiosis, cell suspension, rodent, cell culture, animal model
JoVE Clinical and Translational Medicine
1Department of Pathology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, 2Department of Neurology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine
A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.
Published October 3, 2012. Keywords: Medicine, Neuroscience, Physiology, Anatomy, Prion protein, brain, prion disease, insoluble prion protein, oligomer, ultracentrifugation, Western blotting, Sucrose gradient sedimentation, gel filtration
1Genome Plasticity Laboratory, Department of Cancer Biology, The Scripps Research Institute, 2Flow Cytometry Core, The Scripps Research Institute
An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.
Published April 15, 2011. Keywords: Cellular Biology, meiosis, mouse, FACS, purification, testis