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Université Libre de Bruxelles

4 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Soizic Garaud^*1,2, Chunyan Gu-Trantien^*1,2, Jean-Nicolas Lodewyckx^1,2, Anaïs Boisson^1,2, Pushpamali De Silva^1,2, Laurence Buisseret^1,2, Edoardo Migliori^1,2, Myriam Libin³, Céline Naveaux^1,2, Hugues Duvillier^1,4, Karen Willard-Gallo^1,2

¹Molecular Immunology Unit, Université Libre de Bruxelles, ²Institut Jules Bordet, Université Libre de Bruxelles, ³Institut d'Immunologie Médicale, Université Libre de Bruxelles, ⁴Flow Cytometry Core Facility, Université Libre de Bruxelles

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45⁺ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Biology

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Abdulkadir Abakir¹, Lee Wheldon², Andrew D. Johnson³, Patrick Laurent¹, Alexey Ruzov⁴

¹Laboratoire de Neurophysiologie (CP601), ULB Neuroscience Institute (UNI), Université Libre de Bruxelles, ²Medical Molecular Sciences, Centre for Biomolecular Sciences, University of Nottingham, ³School of Life Sciences, University of Nottingham, ⁴Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Genetics

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Ashley H. Ramsawhook^*1, Lara C. Lewis^*1, Maria Eleftheriou¹, Abdulkadir Abakir¹, Paulina Durczak¹, Robert Markus², Seema Rajani², Nicholas R.F. Hannan¹, Beth Coyle³, Alexey Ruzov¹

¹Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, ²School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, ³Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

Biochemistry

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Raphael Dutoit^1,2, Nathalie Brandt², Dany Van Elder¹, Louis Droogmans¹

¹Laboratory of Microbiology, Department of Molecular Biology, Université Libre de Bruxelles, ²Labiris Institut de Recherche

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050_{H60A H307A} variant.