High content imaging of lysosomal phenotypes in iPSC-derived neurons using the Opera Phenix(TM) High-content Screening System v1

This protocol describes the use of the Opera Phenix high-content screening system and its advanced software Harmony 5.1 to image and analyse live and fixed neuronal cell cultures for the measurement and characterization of lysosomal phenotypes. This includes quantifying lysosomal size and number using a lysosome staining reagent, quantifying lysosomal activity using DQ red-BSA uptake and processing in live cells, and quantifying autophagic flux using P62 and LC3 immunofluorescent staining in the presence and absence of the autophagy-lysosomal pathway inhibitor Bafilomycin A1. The aim of this protocol is to create an automated analysis pipeline that can be used to process large volumes of data for any cell culture for the measurement of lysosomal and autophagosomal phenotypes under different treatment conditions. The use of 3 different and complementary approaches allows of a flexible and tailorable protocol with the advantage that any of these 3 analyses can be used separate or in parallel.