Rutgers, The State University of New Jersey 11 articles published in JoVE Developmental Biology Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes Mansour Aboelenain1,2,3, Karen Schindler1,2, Cecilia S. Blengini1,2 1Department of Genetics, Rutgers, The State University of New Jersey, 2Human Genetics Institute of New Jersey, 3Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes. Cancer Research Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line Minh Ma1,2, Saiaditya Badeti1, James K. Kim1, Dongfang Liu1,3 1Department of Pathology, Immunology and Laboratory Medicine, Rutgers-New Jersey Medical School, 2Department of Microbiology, Biochemistry & Molecular Genetics, Public Health Research Institute Center, New Jersey Medical School, Rutgers University, 3Center for Immunity and Inflammation, New Jersey Medical School, Rutgers-The State University of New Jersey Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells. Bioengineering The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection Joseph J. Sherba1, Maria Atzampou1, Hao Lin2, Jerry W. Shan2, David I. Shreiber1, Jeffrey D. Zahn1 1The Department of Biomedical Engineering, Rutgers, The State University of New Jersey, 2The Department of Mechanical and Aerospace Engineering, Rutgers, The State University of New Jersey This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time. Behavior Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice Andrew Dieterich1,2, Christine N. Yohn1, Benjamin Adam Samuels1,2 1Department of Psychology, Behavioral and Systems Neuroscience Area, The State University of New Jersey, 2Graduate Program in Neuroscience, Rutgers, The State University of New Jersey The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females. Environment Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass Shishir P. S. Chundawat1, Ramendra K. Pal1, Chao Zhao1, Timothy Campbell2, Farzaneh Teymouri2, Josh Videto2, Chandra Nielson2, Bradley Wieferich3, Leonardo Sousa3, Bruce E. Dale3, Venkatesh Balan4, Sarvada Chipkar5, Jacob Aguado5, Emily Burke5, Rebecca G. Ong5 1Department of Chemical and Biochemical Engineering, Rutgers-State University of New Jersey, 2Michigan Biotechnology Institute (MBI), 3Department of Chemical Engineering and Materials Science, Michigan State University, 4Engineering Technology Department, Biotechnology Program, College of Technology, University of Houston, 5Department of Chemical Engineering, Michigan Technological University Ammonia fiber expansion (AFEX) is a thermochemical pretreatment technology that can convert lignocellulosic biomass (e.g., corn stover, rice straw, and sugarcane bagasse) into a highly digestible feedstock for both biofuels and animal feed applications. Here, we describe a laboratory-scale method for conducting AFEX pretreatment on lignocellulosic biomass. Immunology and Infection Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria Shamba Gupta1, G. Marcela Rodriguez1 1Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency. Medicine Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis Sundararajan Venkatesh*1, Manju Chauhan*2, Carolyn Suzuki1, Neeraj Chauhan1,2 1Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, 2Public Health Research Institute, Rutgers, The State University of New Jersey Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer. Biology Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System Ying Zhang1, Juan Dong1,2 1The Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, 2The Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells. Immunology and Infection Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH Sarah M. Misenko1, Samuel F. Bunting1 1Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats. Neuroscience A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals David J. Barker1, David H. Root2, Kevin R. Coffey1, Sisi Ma1, Mark O. West1 1Department of Psychology, Rutgers, the State University of New Jersey, 2Neuronal Networks Section, Integrative Neuroscience Research Branch, National Institute on Drug Abuse Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges. Methods for performing this technique and the equipment necessary are described. Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed. Neuroscience Building An Open-source Robotic Stereotaxic Instrument Kevin R. Coffey1, David J. Barker1, Sisi Ma1, Mark O. West1 1Department of Psychology, Rutgers, The State University of New Jersey This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).
