Walter and Eliza Hall Institute of Medical Research View Institution's Website 7 articles published in JoVE Medicine Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients Sylvia F. Boj1, Annelotte M. Vonk2, Marvin Statia1, Jinyi Su1, Johanna F. Dekkers3, Robert R. G. Vries1, Jeffrey M. Beekman2, Hans Clevers1,4 1Foundation Hubrecht Organoid Technology, 2Department of Pediatric Pulmonology, Regenerative Medicine Centre Utrecht, Wilhelmina Children's Hospital, University Medical Centre Utrecht, 3Department of Stem Cells and Cancer, Walter and Eliza Hall Institute of Medical Research, 4Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators. Medicine Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice Matthias Ernst1,2,3, Adele Preaudet1, Tracy Putoczki1,2 1The Walter and Eliza Hall Institute for Medical Research, 2The Department of Medical Biology, University of Melbourne, 3Olivia Newton-John Cancer Research Institute We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse. Immunology and Infection High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays Danika L. Hill1,2, Emily M. Eriksson1,2, Louis Schofield1,2 1Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, 2Department of Medical Biology, University of Melbourne Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry. Bioengineering A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL) Joseph Dragavon1, Carolyn Sinow2, Alexandra D. Holland1, Abdessalem Rekiki1, Ioanna Theodorou3, Chelsea Samson4, Samantha Blazquez1, Kelly L. Rogers5, Régis Tournebize1,6,7, Spencer L. Shorte1 1Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 2Department of Radiation Oncology, Stanford School of Medicine, 3 Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions. Chemistry Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies Pooja Sharma1,2, Mariam Kaywan-Lutfi1, Logesvaran Krshnan1,2, Eamon F. X. Byrne1,2, Melissa Joy Call1,2, Matthew Edwin Call1,2 1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications. Immunology and Infection Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice Victoria Ryg-Cornejo1, Lisa J. Ioannidis1, Diana S. Hansen1 1The Walter and Eliza Hall Institute of Medical Research A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry. Immunology and Infection Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice Kirsty R. Short1, Dimitri A. Diavatopoulos2, Patrick C. Reading1, Lorena E. Brown1, Kelly L. Rogers3, Richard A. Strugnell1, Odilia L.C. Wijburg1 1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.