Poster Presentation 11th International Symposium on Autophagy 2025

A novel pH-sensitive probe to quantify autophagy on high throughput/content imaging platforms  (#225)

Joe D Ciccotosto 1 , Metta Jana 2 , Lorenzo Galluzzi 3 , Michael Lazarou 4 , David L Hare 1 , Peter J Wookey 1
  1. Department of Medicine-Austin, University of Melbourne, Heidelberg, VIC, Australia
  2. Sir Peter MacCallum Department of Oncology,, Peter MacCallum Cancer Centre, Parkville, Victoria 3052, Australia
  3. Fox Chase Cancer Center,, Philadelphia,, PA,, USA
  4. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia

Background             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 development of novel screening approaches, amenable to high throughput/content applications.

Aim    We devised a simple method to detect autophagy in cultured mammalian cells based on the pH-sensitive fluorescent conjugate CalRexinTM:pHrodoTM Red and high throughput/content imaging.

Methods 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. Cellular responses were imaged with the novel CalRexinTM:pHrodoTM Red (CalRexinTM, Apop Biosciences Pty Ltd) reagent on the Operetta high content screening platform. Co-localisation with pixel definition between CalRexinTM:pHrodoTM Red and markers of early and late endosomes and autolysosomes was studied with high resolution confocal microscopy and Fiji software.

Results          In HeLa cells, the accumulation of CalRexinTM:pHrodoTM Red as elicited by canonical autophagy activators rapamycin, torin-1 or serum starvation and was suppressed by the conventional inhibitors 3-methyladenine, chloroquine, bafilomycin and MRT68921. Similarly, RB1CC1-/- (FIP200) as well as ATG5-/- HeLa cells (which bear genetic defects in two different steps of autophagy) were unable to accumulate CalRexinTM:pHrodoTM Red upon exposure to autophagy activators. Pixel co-localization analysis demonstrated significant overlap between CalRexinTM:pHrodoTM Red and markers of early and late endosomes, as well as LysoTrackerTM. a marker of autolysosomes.

Conclusions            CalRexinTM:pHrodoTM Red is taken up by the endosome-amphisome-autolysosome (low pH ~4.5) pathway, which is connected to canonical autophagy, culminating with a bright red fluorescence. CalRexinTM:pHrodoTM Red provides a novel approach for imaging autophagy on high throughput/content platforms to screen libraries for the identification of novel autophagy-targeting agents.