BloodCenter of Wisconsin View Institution's Website 8 articles published in JoVE Biochemistry Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo Patrick Moran1,2, Yichen Guo3,4, Rong Yuan1,3, Nicholas Barnekow1, Jordan Palmer2, Adam Beck3, Bin Ren3,4,5 1Blood Research Institute, Blood Center of Wisconsin, 2Department of Medicine, Medical College of Wisconsin, 3Department of Surgery, The University of Alabama at Birmingham, 4Department of Biomedical Engineering, The University of Alabama at Birmingham, 5GBS Program, Graduate School, The University of Alabama at Birmingham We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification. Cancer Research Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays Anagha Deshpande1, Bo Rui Chen1, Luyi Zhao1, Kelsey Saddoris1, Mayuri Kerr1, Nan Zhu2, Prashant Mali3, Aniruddha J. Deshpande1 1Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, 2Blood Research Institute, Blood Center of Wisconsin, 3Department of Bioengineering, University of California, San Diego This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well. Immunology and Infection Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells Arash Nanbakhsh1, Brad Best2, Matthew Riese3, Sridhar Rao4, Li Wang5, Jeffrey Medin6, Monica S. Thakar6, Subramaniam Malarkannan1,5,6,7 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, The Blood Center of Wisconsin, 2Vector Core Lab, Blood Research Institute, The Blood Center of Wisconsin, 3Laboratory of Lymphocyte Biology, Blood Research Institute, The Blood Center of Wisconsin, 4Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute, The Blood Center of Wisconsin, 5Department of Microbiology and Immunology, The Medical College of Wisconsin, 6Department of Pediatrics, The Medical College of Wisconsin, 7Department of Medicine, The Medical College of Wisconsin The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation. Immunology and Infection High-throughput Detection Method for Influenza Virus Pawan Kumar1, Allison E. Bartoszek1, Thomas M. Moran2, Jack Gorski3, Sanjib Bhattacharyya4, Jose F. Navidad4, Monica S. Thakar1,5, Subramaniam Malarkannan1,6 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens. Biology Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice Magdalena Sobczak1, Jillian Dargatz1, Magdalena Chrzanowska-Wodnicka1 1Blood Research Institute, BloodCenter of Wisconsin Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin. Immunology and Infection Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS) Sreemanti Basu1, Hope M. Campbell1, Bonnie N. Dittel1, Avijit Ray1 1Blood Research Institute, BloodCenter of Wisconsin For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations. Immunology and Infection Depletion of Specific Cell Populations by Complement Depletion Bonnie N. Dittel1 1BloodCenter of Wisconsin, Blood Research Institute To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity. Immunology and Infection Isolation of Mouse Peritoneal Cavity Cells Avijit Ray1, Bonnie N. Dittel1 1BloodCenter of Wisconsin, Blood Research Institute The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.