Ospedale Pediatrico Bambino Gesù, IRCCS

3 ARTICLES PUBLISHED IN JoVE

Medicine

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Maria Vinci^1,2, Carol Box², Suzanne A. Eccles²

¹Division of Molecular Pathology, The Institute of Cancer Research, ²Division of Cancer Therapeutics, The Institute of Cancer Research

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Biology

Measuring the Confluence of iPSCs Using an Automated Imaging System

Valentina Magliocca^1,2, Maria Vinci³, Tiziana Persichini², Franco Locatelli³, Marco Tartaglia¹, Claudia Compagnucci¹

¹Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, ²Department of Science, University Roma Tre, ³Department of Onco-hematology, Gene and Cell Therapy, Ospedale Pediatrico Bambino Gesù, IRCCS

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Biochemistry

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Giulia Fasano¹, Valeria Bonavolontà², Catia Pedalino², Martina Venditti², Graziamaria Paradisi², Stefania Petrini³, Marco Tartaglia², Antonella Lauri²

¹Research laboratories, Ospedale Pediatrico Bambino Gesù, IRCCS, ²Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, ³Microscopy facility, Research laboratories, Ospedale Pediatrico Bambino Gesù, IRCCS

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.