A novel pH-sensitive probe to quantify autophagy on high throughput/content imaging platforms

Autophagy is deregulated in various pathological conditions including cardiovascular, neurological, neoplastic, autoimmune and degenerative disorders. However, clinically relevant pharmacological modulators of autophagy remain elusive, calling for the development of novel screening approaches, amenable to high throughput/content applications. Cellular responses were imaged with the novel CalRexin TM :pHrodo TM Red (CalRexin TM , Apop Biosciences Pty Ltd) reagent on the Operetta high content screening platform. Human cervical carcinoma HeLa cells, and mutants RB1CC1-/- (FIP200) and ATG5-/- were induced for autophagy with rapamycin, torin-1 and serum deprivation and inhibited with 3-methyladenine, MRT68921, chloroquine and bafilomycin A1. The co-localisation with pixel definition between CalRexin TM :pHrodo TM Red and markers of early and late endosomes and autolysosomes was studied with high resolution confocal microscopy and Fiji software. Pixel co-localization analysis demonstrated significant overlap between CalRexin TM :pHrodo TM Red and markers of early and late endosomes, as well as LysoTracker TM , a marker of autolysosomes.CalRexin TM :pHrodo TM Red is accumulated via the endosome-amphisome-autolysosome (low pH ~4.5) pathway, thus connecting to canonical autophagy and yielding a bright fluorescent signal. CalRexin TM :pHrodo TM Red provides a novel approach for imaging autophagy on high throughput/content platforms and is adaptable for screening large chemical libraries for the identification of novel autophagy-modulators as potential drugs for treatment of chronic diseases.